Recently published data have provided evidence that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) are signaling intermediates in the pathway through which muscle contraction stimulates glucose transport. As exercise promotes both NO production and calcium flux, we examined the relationships between NO-stimulated glucose uptake and calcium-, contraction-, and phosphatidylinositol-3-kinase (PI-3-K)-mediated glucose transport in the isolated incubated rat epitrochlearis muscle preparation. The NO donor sodium nitroprusside (SNP; 10 mmol/1) and dibutyryl cGMP (100 μmol/l) accelerated epitrochlearis glucose transport four- to fivefold above basal levels (P < 0.001) in a manner similar to in vitro contractile activity and the calcium releasing agent N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W7; 100 μmol/1). In the case of SNP, this effect could be completely attributed to an increase in cell surface GLUT4. The effect of SNP on glucose transport was not inhibitable by either wortmannin (1.5 μmol/1) or dantrolene (12.5 μmol/1). Similarly, neither calcium nor contraction stimulation of glucose transport was affected by the NO synthase inhibitors N^G-monomethyl-L-arginine (L-NMMA; 100 μmol/1) or 7-nitroindazole (1 mmol/1). Furthermore, whereas SNP raised epitrochlearis cGMP levels tenfold (P < 0.001), neither in vitro contractile activity nor W7 significantly elevated cGMP. These results indicate that NO/cGMP can markedly stimulate skeletal muscle glucose transport by increasing GLUT4 levels at the cell surface by a mechanism that does not depend on activation of PI-3-K. In addition, since calcium/contraction-stimulated glucose transport is not blocked by NO synthase inhibition and did not elevate cGMP, NO/cGMP may be part of a novel pathway that is distinct from both the insulin- and contraction-activated mechanisms.