Peripheral blood B lymphocytes have been isolated from healthy individuals who were immunized with lymphocytes from HLA-incompatible donors and transformed with Epstein-Barr virus to produce human monoclonal cell lines specific for human HLA molecules. The cell lines have been previously characterized and are known to bind to various class I and class II alloantigens. In this report we describe the molecular characterization of the heavy and light chain variable region gene segments that are utilized by these monoclonal antibodies. Using the polymerase chain reaction and primer pairs specific for the respective constant region and VH or VL family, rearranged variable region gene segments were amplified from cDNA from individual cell lines. Products were then subcloned, sequenced and analysed for gene usage and apparent somatic mutation. The results show that the VH3 gene family predominates in a group of six heavy chains (four out of six) with one VH1 and one VH4 gene segment. The light chain variable region gene family usage is more diverse with 2 V kappa 3, 1 V kappa 1, 2 V lambda 2 and 1 V lambda 3. The extent of apparent somatic mutation is minimal, relative to our previous observations in a group of high affinity human monoclonal antibodies specific for pathogenic organisms.