We have recently shown that the semi-continuous cultivation of a mouse hybridoma line in spinner flasks, with a basal defined medium (BDM) devoid of serum and protein, increases the secretion of the immunoreactive monoclonal antibody (MAb) by a factor of ca. 2.4, compared to culture in flasks with serum-containing medium (Schneider, 1989). To further optimise MAb production, we have now investigated the composition of BDM and the mode of cultivation. Hybridoma cells were inoculated at 0.3 x 10(6) cells/ml in 200 ml of BDM containing 4, 6 or 8 mM glutamine; after 3-4 days (when the cell density reached greater than or equal to 10(6) cells/ml) 20, 40, 60 or 100% of the culture medium were replaced daily by fresh nutritive BDM with or without 33, 66 or 100% cell recycling. Daily, over a total period of 15-16 days, viable cells were counted and the concentration of the MAb, of glucose and glutamine (the main nutrients) and of lactate, alanine and ammonia (the main metabolites) were assayed. These experiments indicate that to provide optimal cell growth and MAb production: (i) the concentration of glutamine in the nutritive medium should be maintained at 8 mM, otherwise it falls on some days to 0 with a significant amount of cell death; at 25 mM, glucose concentration is not a limiting factor, whatever the culture conditions; (ii) with a semi-continuous mode of cultivation, a perfusion rate of 40%/day appears optimal; without cell recycling, a rate of 20% does not provide enough nutrients and/or does not remove enough metabolites, whereas a rate of 60% washes over the cells; with cell recycling, rates of 60 (moderately) or 100% (considerably) increase cellular metabolism without concurrent augmentation of MAb secretion; (iii) the recycling of the cells increases the mean cell densities and the rate of production of the MAb, as well as the rate of consumption of nutrients and of production of metabolites; recycling of 33-66% is optimal, since the total recycling progressively raises the number of dead cells and debris; (iv) there are maximal values for cell densities (ca. 2.5 X 10(6) cells/ml) and MAb production (ca. 26 micrograms/10(6) cells X day) as well as for nutrient consumption and metabolite production (except with a very high perfusion rate).(ABSTRACT TRUNCATED AT 400 WORDS)