We have developed an improved in situ hybridization (ISH) technique for the analysis of human immunoglobulin heavy chain variable (V(H)) gene family expression in suspensions of human B lymphocytes. Oligonucleotide probes specific for framework region (FR) consensus germline sequences for each of the seven human V(H) gene families were designed and hybridization conditions were developed to accommodate the greatest degree of V(H) gene variation, maximize the sensitivity of transcript detection, and assure the specificity of the technique. The hybridization parameters were rigorously characterized by Southern hybridization to a panel of 30 V(H) cDNA clones and by ISH to 17 B cell lines expressing characterized V(H) genes. Results obtained with ISH using V(H) gene family and isotype-specific gene probes correlated well with histochemical measures of Ig gene product expression. Profiles of cellular V(H) gene expression were generated for mitogen stimulated peripheral blood B lymphocytes from six normal subjects. When compared with estimates of frequency of V(H) genes in the human germline, the results were consistent with a random pattern of V(H) family utilization.