The insulin-responsive glucose transporter (GLUT4) is expressed at high levels in fat and skeletal muscle, which account for the majority of insulin-stimulated glucose uptake. However, GLUT4 is also expressed at lower levels in kidney and several other tissues. We have used a variety of protein and mRNA detection techniques to determine the sites of renal GLUT4 expression. Indirect immunofluorescence experiments with two specific anti-peptide antisera detected GLUT4 in the smooth muscle cells of the rat renal microvasculature, in renal glomerulus, and in cultured glomerular mesangial and epithelial cells. PCR amplification of cDNA derived from microdissected renal glomeruli, microvessels and tubules corroborated this distribution of GLUT4, and Northern blotting demonstrated GLUT4 mRNA in cultured glomerular mesangial cells. Both the immunofluorescence and PCR data suggested that GLUT4 is most highly expressed in renal microvessels. Our results show that certain renal cells, such as renal microvascular smooth muscle cells, express the insulin-responsive glucose transporter and therefore may demonstrate altered glucose uptake and metabolism in diabetes mellitus.