Hybridoma cells usually grow to fairly low cell densities in batch cultures (1-3 x 10(6) cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1-3 x 10(5) cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.