The long-term in vitro effects of insulin on insulin binding and glucose transport were studied using rat adipocytes in a time-dependent manner. Isolated fat cells were incubated with insulin (100 ng/ml) for 4, 8 and 24 h in a TCM 199 medium, at 37 degrees C, and then insulin binding (37 degrees C, 60 min) and 3-O-methylglucose transport (37 degrees C, 2 s) were determined. Decreased insulin binding was demonstrated in the cells incubated with insulin for 8 h, and Scatchard analysis revealed that receptor number was decreased to 61.7% of that of control cells. Thus, insulin-induced down regulation of receptors was evident after 8 h incubation with insulin. On the other hand, 8 h incubation with insulin resulted in markedly increased basal (i.e., in the absence of insulin) glucose transport up to 246% of control values. In the cells incubated with insulin for 24 h, maximally insulin-stimulated glucose transport was significantly increased up to 248% of control value. Thus, these results suggested that insulin-induced down regulation of receptors appeared to be coupled with increased cell-surface glucose transporters, and that there was a time-lag between down regulation of insulin receptors and increase of available glucose transporters in the plasma membrane.