During pregnancy, islets undergo a number of up-regulatory changes to meet the increased need for insulin. One of the most important changes is an increase in glucose-stimulated insulin secretion with a reduction in the glucose-stimulation threshold. Similarly, placental lactogen and PRL induce the same changes in islets as pregnancy. In this study, we examined the effects of pregnancy and PRL treatment of islets in vitro on insulin secretion; glucokinase and hexokinase activities; glucokinase, hexokinase, and glucose trans porter 2 protein levels; and rates of glucose utilization and oxidation. Glucokinase activity was 4.9 ± 0.4 pmol glucose/ng DNA·h in control islets and was significantly increased by 50% in islets on day 15 of pregnancy and by 60% on day 20 of pregnancy. Hexokinase activity was 11.7 ± 0.9 pmol glucose/ng DNA·h in control islets and was increased by 20% in islets on day 15 of pregnancy and by 90% on day 20 of pregnancy. In the in vitro studies, glucokinase activity was 7.4 ± 0.89 pmol glucose/ng DNA·h in control islets. PRL treatment of islets in vitro increased glucokinase activity by 60%, an effect similar to that observed in the pregnancy islets. In contrast, hexokinase activity was nearly undetectable in cultured islets, whether control or PRL treated. Quantitative Western blot analysis of glucokinase and hexokinase was performed using equivalent amounts of protein per lane for all experimental groups. On a protein equivalency basis, glucokinase expression levels were the same in control islets and islets on days 15 and 20 of pregnancy. Likewise, hexokinase levels were not different between control islets and islets on days 15 and 20 of pregnancy. Similarly, Western blot analysis of cultured islets indicated that there was no effect of PRL on glucokinase or hexokinase levels. However, when enzyme levels were normalized on the basis of DNA, the levels of expression appeared to be commensurate with their activities. In cultured islets, the very low level of hexokinase activity corresponded to the low level of hexokinase detected by the Western blots. Glucose transporter 2, as determined by Western blot quantification, was increased 2 fold in pregnancy islets on day 15 and increased by 45% in pregnancy islets on day 20. Similar results were observed in cultured islets where glucose transporter 2 was increased 2-fold in PRL- treated islets. Islet glucose utilization and oxidation rates on day 15 of pregnancy were significantly greater than those in control islets at all glucose concentrations examined. This enhanced glucose sensitivity resulted in a shill of the glucose utilization and oxidation response curves to the left. Comparable results were obtained from islets on day 20 of pregnancy. PRL treatment of islets in vitro resulted in the same changes in glucose utilization and oxidation rates that were observed during pregnancy. These results demonstrate changes in glucokinase, hexokinase, and glucose transporter 2 levels and glucose metabolism that occur as islets adapt to an increased need for insulin secretion during pregnancy. The results also indicate that these same changes can be induced by PRL treatment of islets in vitro. This provides further evidence that the long term adaptive changes that occur under the normoglycemic conditions of pregnancy are mediated by lactogen-regulated events.