Photoaffinity labeling of atrial natriuretic factor (ANF) receptor in the plasma membranes from bovine aortic smooth muscle tissue using Nα5-(4-azidobenzoyl)-ANF-(5-28)-peptide labeled with 125I yielded a 130-kDa band. However, when smooth muscle cells from the same bovine aorta were placed in culture, the 130-kDa receptor quickly disappeared and a 60-kDa band began to appear at high density. After three passages, essentially no 130-kDa band was found and only the 60-kDa band was strongly labeled. The primary structures of the two receptor forms were compared by radiochemical peptide mapping after endoproteinase Glu-C digestion of photoaffinity-labeled and detergent-solubilized 130-kDa receptor from the aorta or the 60-kDa receptor from the cultured cells. The peptide mapping showed courses of digestion that were significantly different from each other, suggesting difference in their primary structures. The basal guanylate cyclase activity in the aortic membranes was 1.0 pmol cGMP produced · min−1· mg protein−1 at 37°C using Mn2+-GTP as substrate. The corresponding activity in the membranes from the cultured cells was 20 fmol cGMP · min−1· mg protein−1· Binding studies gave a density of binding sites (Bmax) of 82 fmol/mg protein for the aortic membranes and 850 fmol/mg protein for the cultured cell membranes. These data suggest that the major form of ANF receptor in the cultured cells, namely the 60-kDa receptor, lacked guanylate cyclase activity. Northern blot analysis of poly(A)-RNA extracted form bovine thoracic aorta or adrenal cortex gave a single 3.6-kb band when 32P-labeled human A-type ANF receptor cDNA was used as a hybridization probe. However, no band was detected when C-receptor cDNA was used as a probe. In addition to the major 130-kDa band, extended SDS/PAGE revealed two additional faint bands with estimated molecular masses of 126 kDa and 135 kDa. Treatment with endoglycosidase H resulted in disappearance of the 126-kDa band and appearance of a 100-kDa band. The 130-kDa and 135-kDa bands were unchanged. Treatment by endoglycosidase F or glycopeptidase F reduced all three bands to a single 100-kDa band. These results suggest that the slight difference in mobility is due to different states of glycosylation. Competitive protection experiments showed binding specificity in the order of ANF > BNP ≫ CNP, AP-I for all three bands, indicating that the state of glycosylation had no effect on the ligand specificity. Photoaffinity labeling of bovine adrenal cortex membranes gave a single 130-kDa band. The same experiment with lung membranes yielded a 130-kDa band and a 60-kDa band at about 1: 1 ratio. The data presented in this report indicate that the vascular smooth muscle contains predominantly the 130-kDa guanylate-cyclase-coupled ANF receptor in vivo, and that C-receptors found in cultured vascular smooth muscle cells have been induced artificially from cell culturing.