Murine bone marrow-derived macrophages (BMDM), a homogeneous cell population easily obtainable in large quantities and at reproducible quality by in vitro differentiation, were used as feeder cells for human B cell hybridomas after fusion or during recloning. We used as antigens for the in vitro immunization of human B lymphocytes from peripheral blood as well as from tonsils: (i) synthetic peptides representing immunogenic sequences of gp160 and Nef of HIV-1, coupled to the lipopeptide carrier N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2 RS)-propyl]-(R)-cysteinyl(-seryl-seryl) (P3 CSS-[gp160(303-329)] and P3C-nef24), (ii) the toxins saxitoxin and microcystin, coupled to BSA (BSA-STX and BSA-MCYST). After fusion with the mouse-human heteromyeloma CB-F7, we could demonstrate that BMDM exert a strong growth supporting effect on post-fusion cultures, resulting in 81.6% versus 23.6% growth-positive wells for P3C-nef24, and 100% versus 71.2% growth-positive wells for BSA-STX stimulated cells in cultures with and without BMDM, respectively. Furthermore, clones in wells with BMDM grew much more rapidly, resulting in 24.3% versus 3.6%, 98.1% versus 42.2% and 56.7% versus 6.7% of cultures ready for screening 2 weeks after fusion of P3C-nef24, P3CSS-[gp160(303-329)], and BSA-STX stimulated lymphocytes, respectively. Apart from their effect on cell growth, murine BMDM also increased the percentage of immunoglobulin (Ig)-producing cultures after fusion, as shown for BSA-STX stimulated lymphocytes (47.8% versus 6.7%), as well as the percentage of cultures producing specific antibodies, as demonstrated with BSA-MCYST activated cells (42% versus 10%). Finally, recloning efficiencies of two human B cell hybridomas (E 10 and F 2) were raised profoundly by BMDM, resulting in 100% versus 64.2% and 90.9% versus 44.2% growth-positive wells after recloning on a ten cells/well level. As murine BMDM can also be stored in liquid nitrogen without loss of activity, they constitute ideal feeder cells for the establishment of human B cell hybridomas.