A family of cell surface glycoproteins with a molecular weight of 96,000 (gp96) has recently been implicated in the individually distinct immunogenicity of chemically induced sarcomas of inbred mice. Rabbit antiserum to murine gp96 detects an antigenically related Mr 96,000 cell surface glycoprotein on two cultured human melanoma cell lines, SK-MEL-13 and SK-MEL-177. Molecular probes for 5' and 3' ends of the murine gp96 gene detect a 3.5-kilobase transcript in RNA preparations from melanoma cells, similar to the murine gp96 transcript. While 5' probes do not hybridize to Southern blots of genomic human DNA, the 3' probes identify several distinct bands under stringent hybridization and washing conditions. This suggests that the 3' end of the gp96 gene is more conserved than its 5' end. No gross alterations in gp96 gene organization were detected in melanoma cells. B-lymphoblastoid cell lines derived from four different individuals also showed no restriction fragment polymorphism in the gp96 gene.