Insulin and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) stimulatory effects on adipocyte glucose transport were compared for their sensitivity to: (1) sphingosine and staurosporine, two potent protein kinase C (PKC) inhibitors; and (2) phenylarsine oxide (PhAsO), a dithiol reagent blocking insulin-stimulated glucose transport. None affected basal 2-deoxyglucose transport, cell viability, cellular ATP content, or insulin binding. Insulin- and PMA-stimulated 2-deoxyglucose transport were both markedly inhibited by sphingosine (5-50 microM) and staurosporine (0.1-2 microM), although with differences in the extents of maximal inhibitions (65 and 48% vs. 88 and 98%) and the concentrations of the drugs causing the half-maximal inhibitions observed in the experiments (2- to 3-fold higher for insulin). Insulin and PMA both altered PKC along with glucose transport, either by increasing its activity in the cytosol or by promoting its translocation to membrane. Insulin- and PMA-stimulated 2-deoxyglucose transport were both inhibited selectively by PhAsO (0.1-1 microM), at almost identical maximal inhibitions (84 and 90%) and IC50 values (0.18 and 0.16 microM). Furthermore, insulin- and PMA-induced increases in transport Vmax (6.5- and 3.4-fold) were both reduced by 89% by PhAsO, which, however, failed to affect the decrease in transport Km (1.7-fold) exclusively induced by insulin. Likewise, PhAsO did not affect insulin or PMA activation of PKC. The results suggest that insulin activates adipocyte glucose transport through: (1) a PKC-dependent mechanism requiring cellular dithiols, responsible for a part of the hormone-induced increase in transport Vmax; and (2) a PKC-independent mechanism responsible for both a further increase in transport Vmax and a decrease in transport Km.