In vitro effects of bovine somatotropin (bST) and insulin plus dexamethasone on lipolysis were evaluated using chronic cultures (48 h) of adipose tissue from lactating cows. Treatments were control (culture medium alone), bST (100 ng/ml), insulin (100 ng/ml) plus dexamethasome (10 nM), and insulin plus dexamethasone plus bST. Following the 48-h cultures, rates of lipolysis were measured in 3-h incubations with isoproterenol (10 microM), adenosine deaminase (0.75 U/ml), and various concentrations of a nonhydrolyzable adenosine analog. The addition of bST to cultures did not alter basal or isoproterenol-stimulated lipolysis. However, the ability of adenosine to inhibit rates of lipolysis was reduced by bST. When measured in the presence of maximal concentrations of adenosine analog, isoproterenol caused an increase in lipolysis above basal, which was twofold greater for explants cultured with insulin plus dexamethasone plus bST than for explants cultured with insulin plus dexamethasone. Dose-response curves for adenosine inhibition of isoproterenol-stimulated lipolysis demonstrated that chronic culture with bST decreased adipose tissue responsiveness and sensitivity to adenosine. Overall, results demonstrated that an in vitro chronic culture system can be used to examine factors that regulate lipolysis. The addition of insulin plus dexamethasone to chronic cultures better maintained the intracellular signaling system, including sensitivity and responsiveness to adenosine inhibition of lipolysis. Results also confirm that bST alters the antilipolytic response to adenosine. Thus, bST effects are in large part due to a relief in the tonic inhibition of lipolysis via changes in the inhibitory G-protein signaling system.