Caveolin, an integral membrane protein, is a principal component of caveolae membranes in vivo. Two isoforms of caveolin have been identified: a slower migrating 24-kDa species (alpha-isoform) and a faster migrating 21-kDa species (beta-isoform). Little is known about how these isoforms differ, either structurally or functionally. Here we have begun to study the differences between these two isoforms. Microsequencing of caveolin reveals that both isoforms contain internal caveolin residues 47-77. In a second independent approach, we recombinantly expressed caveolin in a caveolin-negative cell line (FRT cells). Stable transfection of FRT cells with the full-length caveolin cDNA resulted in the expression of both caveolin isoforms, indicating that they can be derived from a single cDNA. Using extracts from caveolin-expressing FRT cells, we fortuitously identified a monoclonal antibody that recognizes only the alpha-isoform of caveolin. Epitope mapping of this monoclonal antibody reveals that it recognizes an epitope within the extreme N terminus of caveolin, specifically residues 1-21. These results suggest that alpha- and beta-isoforms of caveolin differ in their N-terminal protein sequences. To independently evaluate this possibility, we placed an epitope tag at either the extreme N or C terminus of full-length caveolin. Results of these "tagging" experiments clearly demonstrate that (i) both isoforms of caveolin contain a complete C terminus and (ii) that the alpha-isoform contains a complete N terminus while the beta-isoform lacks N-terminal-specific protein sequences. Mutational analysis reveals that these two isoforms apparently derive from the use of two alternate start sites: methionine at position 1 and an internal methionine at position 32. This would explain the approximately 3-kDa difference in their apparent migration in SDS-polyacrylamide electrophoresis gels. In addition, using isoform-specific antibody probes we show that caveolin isoforms may assume a distinct but overlapping subcellular distribution by confocal immunofluorescence microscopy. We discuss the possible implications of these differences between alpha- and beta-caveolin.