Plasma membranes prepared from pig liver incubated with insulin (50-300 microU/ml) resulted in the release of at least two insulin chemical mediators. They appeared to modulate the activity of adenylate cyclase in liver plasma membranes of pig. One of them was fraction 1 of insulin mediator (M. W. about 3700-4000 dalton) which markedly stimulated the activity of the enzyme, the other was fraction 2 of insulin mediator (M. W. about 1000 dalton) which inhibited the enzyme activity. The results showed that the inhibitor of fraction 2 generated was significantly higher than that of fraction 1 when the membranes were incubated with insulin of low concentration (50-100 microU/ml). On the other hand, the generation of stimulator of fraction 1 from plasma membranes incubated with insulin of high concentration (200 microU/ml) was higher than that of fraction 2. So the ratio of yield between two mediators produced from the membranes was dependent on the concentration of insulin added. The results also showed that the effect of fraction 1 of insulin mediator on adenylate cyclase activity in liver cell plasma membranes was biphasic while fraction 2 of insulin mediator showed an inhibitory effect only even though it was at very high concentration. The results that both mediators combined with Gpp(NH)p and forskolin to affect the enzyme activity show that the action of insulin mediators likely resides in the GTP regulatory component of adenylate cyclase.