Activated human mononuclear cells (MCs) were coincubated for 8 h with HIT cells, a clonal cell line of pancreatic islet β-cells. Measurements of HIT cell viability and insulin secretion were determined to 1) ascertain whether activated MCs can alter β-cell viability in the absence of exogenously provided cytokines, 2) examine this response over a range of MC-HIT cell ratios, and 3) identify mechanisms responsible for altered insulin release consequent to MC-induced HIT cell damage. HIT cell viability was markedly decreased by activated MCs during an 8-h coincubation. HIT cell lysis could be attributed to activated natural killer cells, and lysis did not occur in the presence of activated T-lymphocyte clones. Activated MCs caused a marked early increase in insulin release from HIT cells (increase at 2 h: 7.75 ± 0.16 nM for activated MCs, 2.66 ± 0.09 nM for control; P < 0.001). Insulin levels by the 8th h of the coincubation were significantly lower than the 2-h peak (4.33 ± 0.13 vs. 7.75 ± 0.16 nM, P < 0.001). These changes in insulin were dependent on the ratio of activated MCs to HIT cells with the effects clearly evident at an activated MC-HIT cell ratio of ≥10:1. Pretreatment of activated MCs and HIT cells with prostaglandin-synthesis inhibitors did not prevent the cytotoxic effects of activated MCs on HIT cells. Somatostatin did not inhibit the early exaggerated insulin release, suggesting that these increased insulin levels represented leakage of insulin from damaged HIT cells rather than functional insulin secretion. The decrements in insulin levels during the later stages of the experiments were determined by high-performance liquid chromatography to be caused by degradation of previously released insulin. These studies establish that activated MCs cause HIT cell lysis in the absence of exogenously provided cytokines and that HIT cells can be used as targets to examine the functional consequences of exposing pancreatic islet β-cells to activated MCs.