We have investigated antibody production in 3T3 filler cell-containing murine B lymphocyte cultures, stimulated with LPS and an IL-4-containing lymphokine mixture. At low cell density cultures produced 1.8 +/- 0.6 ng of IgM and 4.2 +/- 1.7 ng of IgG1 per input B cell. It was found that 21.7 +/- 3.5% of spleen cells, or approximately 43% of B cells, produce IgM under these conditions, and 11.9 +/- 5.5% spleen cells, approximately 24% of B cells, produced IgG1. Therefore, the mean IgM production per IgM-positive clone was 4.2 ng, and the mean IgG1 production per IgG1-positive clone was 17.6 ng. A cell density of about 10,000 B cells/ml was found to produce maximal antibody per input cell. A 32-fold increase above the maximum cell density resulted in a 600-fold decrease in IgG1 production per B cell. IgM production was also found to be inhibited above this concentration of cells, but to a six-fold lesser extent. Cell proliferation in dense cultures was also found to be diminished in a cell concentration-dependent manner, partially accounting for the observed inhibition phenomenon. The replenishment of media, LPS and growth factors was able to lessen the inhibition of dense cultures, but not to maximal levels. Overall, this work identified the upper limit of cell density for in vitro cloning of B lymphocytes for isotype switch and repertoire analysis. The most important conclusion is that antibody production is grossly suboptimal at the cell densities frequently used in the literature.