Treatment of saponin-permeabilized adipocytes with NAD enhanced adenylyl cyclase activity stimulated by GT gamma S, [Al/F-4](-), isoproterenol, and forskolin in membrane fractions and potentiated isoproterenol-induced cAMP accumulation in whole cells. In parallel, when permeabilized adipocytes were incubated with [P-32]NAD, there was significant incorporation of [P-32]ADP-ribose in a 44-kDa acceptor membrane protein. This reaction was inhibited by L-arginine and was enhanced by the addition of GTP gamma S. Surprisingly, this 44-kDa protein could not be identified as Gs protein: (1) It was not recognized by Gs alpha specific antibody; (2) it did not comigrate with the major cholera toxin substrates in either 10% SDS-PAGE or two-dimensional electrophoresis; (3) a pretreatment of adipocytes with NAD did not decrease cholera toxin-mediated ADP-ribosylation of Gs alpha proteins on membrane fractions. Our results indicate that NAD did not induce endogenous ADP-ribosylation of Gs alpha in permeabilized rat adipocytes but nonetheless modified the adenylylcyclase response.