The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.