Exposure of Clone 9 cells, a nontransformed rat liver cell line expressing only the Glutl glucose transporter isoform, to the guanylyl cyclase inhibitor LY-83583 was found to stimulate the rate of glucose transport (approximately 7- to 8-fold in 1 h). A similar response to LY-83583 was found in NIH 3T3 fibroblasts, 3T3-L1 pre-adipocytes, and C2C12 myoblasts. Neither the rate of glucose transport in cells under control conditions nor the effect of LY-83583 on glucose transport was altered by 10, 50, or 100 microM 8-bromo-cGMP or by addition of cGMP phosphodiesterase inhibitors, zaprinast, or dipyridamole suggesting that glucose transport and the response to LY-83583 is independent of cGMP levels. In addition, the effect of LY-83583 on glucose transport was not mediated by inhibition of oxidative phosphorylation, since exposure to the agent resulted in no increase in lactate production. Incubation of Clone 9 cells in the presence of the phospholipase C inhibitor U73122, however, attenuated the glucose transport response to LY-83583. Moreover, exposure to LY-83583 resulted in a rise in cell diacylglycerol content, and preincubation with U73122 significantly diminished this rise as well as the glucose transport response to LY-83583. The stimulatory effect of LY-83583 on glucose transport was significantly blocked by thapsigargin. Down-regulation of protein kinase C activity, resulting from 24 h pre-incubation in the presence of 160 nM phorbol-12-myristate 13-acetate, did not attenuate the glucose transport response to LY-83583. It is concluded that the stimulation of glucose transport in response to LY-83583 is independent of changes in cGMP levels, is not mediated by inhibition of oxidative phosphorylation, and is mediated, at least in part, through stimulation of the phospholipase C pathway.