We have identified two RHAMM (receptor for hyaluronan-mediated motility) isoforms that encode an alternatively spliced exon 4 (Hall, C. L., Yang, B., Yang, X., Zhang, S., Turley, M., Samuel, S., Lange, L. A., Wang, C., Curpen, G. D., Savani, R. C., Greenberg, A. H., and Turley, E. A. (1995) Cell 82, 19-26 and Wang, C., Entwistle, J., Hou, G., Li, Q., and Turley, E. A. (1996) Gene 174, 299-306). One of these, RHAMM variant 4 (RHAMMv4), is transforming when overexpressed and regulates Ras signaling (Hall et al.). Here we note using flow cytometry and confocal analysis that RHAMM isoforms encoding exon 4 occur both on the cell surface and in the cytoplasm. Epitope-tagging experiments indicate that RHAMMv4 occurs only in the cytoplasm. Several observations suggest that both cell surface RHAMM isoforms and RHAMMv4 are involved in regulating extracellular-regulated kinase (ERK) activity. Affinity-purified anti-RHAMM exon 4 antibodies block the ability of platelet-derived growth factor to activate ERK, and these reagents modify the protein tyrosine phosphorylation profile of proteins resulting from treatment with platelet-derived growth factor. A dominant negative form of RHAMMv4 inhibits mutant active Ras activation of ERK and coimmunoprecipitates with both mitogen-activated protein kinase kinase and ERK, suggesting that the intracellular RHAMMv4 acts downstream of Ras, possibly at the level of mitogen-activated protein kinase kinase-ERK interactions. Consistent with this, overexpression of RHAMMv4 constitutively activates ERK. These results identify a novel mechanism for the regulation of the Ras-ERK signaling pathway and suggest that RHAMM plays multiple roles in this regulation.