Multiple myeloma (MM) is an incurable plasma cell malignancy. To investigate biochemical lesions associated with MM, we constructed an expression cDNA library from the OPM-2 human myeloma line. A highly transforming H-Ras mutant was identified by transfection analysis using NIH 3T3 cells. DNA sequencing demonstrated a single-point mutation at position 117 located in the guanine nucleotide-binding site resulting in a lysine-to-glutamic acid substitution. This mutant, H-Ras (K117E), was found to be constitutively activated in terms of GTP binding. We compared the biological effects of H-Ras (K117E) and H-Ras (G12V) in 32D murine hematopoietic progenitor cells. Whereas both Ras proteins are constitutively activated, 32D cells expressing H-Ras (G12V) are still dependent on IL-3 for survival and proliferation while cells carrying H-Ras (K117E) become IL-3 independent. Similar experiments conducted with the B9 line, an IL-6-dependent hybridoma, also demonstrated that B9/H-Ras (K117E) became IL-6-independent. Expression of H-Ras (K117E) in the human IL-6-dependent ANBL-6 myeloma line resulted in enhanced proliferation at suboptimal concentrations of IL-6. These observations suggest that H-Ras mutations at the binding site for the GTP nucleotide ring structure may also represent activating lesions and have additional biological effects when compared to previously described Ras mutants.