The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.