The lg VH region sequence in 48 patients with multiple myeloma (MM) was analyzed to characterize the malignant cell of origin. The sequences were obtained after amplification of bone marrow cDNA by using VH family-specific and CH primers, then compared with either directly sequenced patient germ-line or published VH gen sequences to assay for somatic mutation. Because somatic hypermutation of the VH gene occurs late in B cell development, its presence has been helpful in determining the cell of origin in other B cell malignancies. Overall, a median of 8.2% of the nucleotides had evidence of substitution within each VH gene sequence (range=2.7% to 16.5%), which is more prevalent than in any other reported tumor type. Strong evidence of prior antigenic selection pressure was also evident. The ratio of nucleotide substitutions that resulted in amino acid replacement was significantly higher in the complementarity-determining region than in the framework region (3.25 vs 1.56, respectively; p < 0.00005). No VH gene intraclonal diversity was noted, despite sequencing multiple clones (3-16) from each patient, nor was there evidence of further VH gene somatic mutation over the course of three patients' disease. These findings strongly imply that the malignant clone in MM evolves from a cell late in B cell development.