The in vitro production of macrophage colony-stimulating factor (M-CSF) was studied in 14 human lymphoblastoid cell lines and their lineages were ascertained by surface phenotype analysis. M-CSF gene transcripts were detected in a T-lymphocyte-derived cell line (CCRF-CEM) and 3 B-lymphoblastoid cell lines (IM-9, BALL-1, and CCRF-SB) by Northern-blot analysis. The secretion of M-CSF protein into the culture supernatant by each cell line was also studied using an enzyme-linked immunosorbent assay for M-CSF. The 4 cell lines which expressed the M-CSF gene secreted considerable amounts of M-CSF into their culture supernatants, while the 10 cell lines without M-CSF gene expression did not do so. The cell lines which constitutively produced M-CSF were then subjected to Southern-blot analysis of the M-CSF gene structure, and all 14 cell lines were examined for infection by the Epstein-Barr virus. Neither structural changes nor amplification of the M-CSF gene were detected, and Epstein-Barr virus infection was found to be not directly related to M-CSF production.