We have previously shown that CD44 partly mediates ovarian cancer cell attachment to peritoneal mesothelium through recognition of mesothelial-associated hyaluronate. CD44 is a major receptor for hyaluronate and exists as a standard 90-180-kDa form (CD44H), as well as several higher molecular mass variant forms produced by alternative splicing. To determine whether functional differences exist between CD44H and its variants we have investigated the relationship between CD44 isoform expression and mesothelial adhesion in 12 ovarian cancer cell lines. Eight lines were CD44 positive (range, 83-94%) and demonstrated significant binding to mesothelium and hyaluronate, whereas two lines showed reduced CD44 levels (3-13%) and demonstrated decreased binding. Interestingly, two other lines (OVC-3 and SW626) expressed CD44 in the majority of cells (>93%) and yet bound weakly to mesothelium. Mean linear fluorescence intensity of CD44 expressed by OVC-3 and SW626 cells was approximately one-half that of strongly binding cell lines, suggesting that the ability to adhere may be partly related to CD44 surface density. However, immunoprecipitation and immunoblot analyses revealed that standard CD44H represented only 23-31% of total CD44 in weakly binding cells, with the majority of species being comprised of CD44 variants. Indirect immunofluorescence of OVC-3 and SW626 cells confirmed the presence of CD44 variants containing exons v3, v6, and v9. In contrast, CD44H represented the majority (75-86%) of total CD44 expressed by strongly binding cell lines such as CAOV-3 and UPN36T. Transfection of CD44H cDNA into weakly binding OVC-3 cells restored significant mesothelial binding which was partly blocked by anti-CD44 antibody. These data suggest that the expression of CD44 is necessary but not sufficient for mediating attachment of ovarian cancer cells to mesothelium. Although CD44 variants may constitute the major CD44 species in certain ovarian cancer cell lines, it appears that these CD44 species are not always capable of mediating significant binding to mesothelium or hyaluronate. Rather, an adequate level of CD44H is the critical determinant of binding in this system. The role of CD44 variants in the process of ovarian cancer metastasis will require further investigation.