Besides their regulatory role in embryogenesis, homeobox (HOX) genes are expressed in a specific manner in hematopoietic cell lineages, implying a role in the molecular regulation of hematopoiesis. Some HOX C cluster genes are found to be expressed in lymphoid cells of mice and humans. Their function and expression in normal hematopoiesis are still largely unknown. We have studied the mRNA expression of HOXC4, HOXC5, and HOXC6 in several stages of lymphocyte maturation by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNA in situ hybridization (RISH). We examined CD34+/CD38low and CD34+/CD38high cells obtained from normal donor bone marrow (BM), a panel of 19 lymphoid cell lines, several types of leukemias and non-Hodgkin's lymphomas (NHL), and lymphocytes isolated from tonsillar tissue and peripheral blood (PB). HOXC4 and HOXC6 were found to be expressed during maturation in B- and T-lymphoid cells. The expression of each gene was found to be initiated at different cell maturation stages. HOXC4 transcripts were present in CD34+/CD38low cells, which are thought to comprise stem cells and noncommitted progenitor cells, and in subsequent stages to terminally maturated lymphoid cells. HOXC6 expression is initiated in equivalents of prothymocyte and pre-pre-B cell stage and remains present in mature cells. However, HOXC5 is only expressed in neoplastic cell lines and in neoplastic cells of NHL, but not in CD34+ BM cells, nor in resting or activated lymphoid cells isolated from tonsil, PB, or in leukemia cells. In cell lines, weak expression of HOXC5 is initiated in equivalents of pre-B cell and common thymocyte stage and is continuously expressed in mature cell lines. Semi-quantitative RT-PCR showed that expression levels of HOXC5 were much lower than those of HOXC4 and HOXC6; furthermore an increase of expression of HOXC4, HOXC5, and HOXC6 during lymphoid cell differentiation was demonstrated. Thus, mainly mature lymphoid cell lines and neoplastic cells of NHL do express HOXC5, in contrast to the lack of expression in normal lymphoid cells and leukemias. These findings suggest involvement of HOXC5 in lymphomagenesis.