We have utilized subtractive hybridization to isolate 16 distinct cDNA sequences representing genes expressed in pre-B-cell lines but not myeloma cell or fibroblast lines. These sequences represent RNA transcripts that vary in abundance in pre-B-cell lines from 0.001% to 0.05%. Five of these sequences were not related to any known genes. One was related to but distinct from known myosin regulatory light chain genes and another encoded a protein with lectin domains. Three represented previously identified genes encoding carbonic anhydrase type II, thymosin, and CD2; these genes were not previously known to be specifically expressed in early stages of B-cell development. Other isolated genes corresponded to pre-B-cell-specific or pre-B-cell/B cell-specific genes recently described by others. The isolated cDNA sequences may be divided into two general categories--those representing genes expressed only in the pre-B-cell stage of B-cell development and those expressed in both the pre-B-cell and B-cell stages. The in vivo expression patterns of the identified genes suggest that some function specifically in lymphocytes while others may have roles in additional lineages.