The activity of the glycogen synthetase interconverting enzymes, synthetase D phosphatase, and synthetase I kinase have been studied in in vitro, perfused rat hearts and compared to the activity in hearts from intact rats. Synthetase D phosphatase activity was the same in both groups when assayed in the presence of mercaptoethanol. In the absence of this sulfhydryl reducing reagent the activity was lower in the perfused hearts. Synthetase I kinase activity in the presence of saturating amounts of 3′,5′-adenosine monophosphate (cyclic AMP) was not greatly different in the perfused and non-perfused hearts but in the absence of cyclic AMP the activity was definitely reduced. This was not due to small molecular activators, inhibitors, or cofactors, but rather appeared to be the result of a change in the enzyme per se. These result suggest that there are two forms of synthetase I kinase which differ in their sensitivity to cyclic AMP. The conversion of the kinase to a more cyclic AMP dependent form by in vitro perfusion alone is similar to the effect produced in skeletal muscle by insulin6. This may be the reason the perfused heart synthetase system does not respond to exogenous insulin administration.