The effects of several calmodulin antagonists on the antilipolytic activity of insulin were studied in isolated rat adipocytes. N-(6-aminohexyl)-5-Chloro-1-naphthalenesulfonamide (W-7) antagonized the inhibitory effect of insulin on lipolysis due to dibutyryl cyclic AMP (Bt2cAMP) (0.5 mM), but not due to adrenaline (1 microM) or 3-isobutyryl-1-methylxanthine (IBMX) (0.1 mM). The concentration of W-7 required for half-maximal suppression of antilipolytic effect of insulin (25 microU/ml) was 12 microM. High concentrations (up to 1000 microU/ml) of insulin could not overcome this suppression by W-7. W-7 did not affect the insulin binding to its receptor on fat cells. Other types of calmodulin antagonists such as trifluoperazine or calmidazolium did not antagonize the inhibitory effect of insulin. These findings show that the insulin receptor or calmodulin may not be involved in this suppressive effect of W-7.