Previously, we have identified an alpha IIb beta 3-like integrin in tumor cells by using antibodies against platelet alpha IIb beta 3. However, alpha IIb beta 3 was considered to be expressed strictly in megakaryocyte lineage cells. In order to resolve this controversy, the alpha IIb beta 3-like integrin in murine B16 amelanotic melanoma (B16a) cells was characterized at DNA, RNA, and protein levels. The presence of alpha 5, alpha v, alpha IIb, beta 1, and beta 3 genes in B16a cells was confirmed by Southern analysis. mRNAs for all these integrins except alpha v were detectable by Northern blotting. The alpha IIb beta 3 protein was identified by Western blotting using subunit-specific antibodies and by immunoprecipitation using complex-specific antibody. The alpha IIb beta 3 integrin was localized intracellularly by immunocytochemistry. Finally, alpha IIb and beta 3 mRNAs were amplified by reverse transcription-polymerase chain reaction and the identity of alpha IIb was verified by sequencing. Partial DNA and deduced amino acid sequence analysis showed that B16a alpha IIb shares approximately 80% homology with the human alpha IIb and approximately 90% homology with the rat alpha IIb, whereas B16a alpha IIb shares only approximately 26% homology with the human alpha v. These experiments indicate that the alpha IIb beta 3-like protein in B16a cells is the authentic alpha IIb beta 3 and demonstrate, for the first time, that integrin alpha IIb beta 3 is not confined to megakaryocyte lineage cells.