The influence of Kupffer cells (KC) from normal, D-galactosamine- and thioacetamide-injured liver on the growth of rat liver fat-storing cells (FSC) in culture was studied. The supplementation of FSC incubation medium with normal and galactosamine-KC-conditioned media for 4 days caused 1.5- and 1.9-fold increase (P less than 0.005), respectively, in the DNA contents of FSC on the fifth day of culture. The stimulant effect reached a maximum at a 1:2 dilution of the conditioned medium with FSC incubation medium. The population doubling time of FSC grown in the absence of conditioned medium was calculated to be 41.5 +/- 3 hr; it was greatly shortened by KC-conditioned medium from galactosamine-treated rats (24 +/- 2 hr), by normal KC-conditioned medium (28 +/- 4 hr), and by KC medium from thioacetamide-injured rats (33 +/- 5 hr). Similarly KC-conditioned media of either source stimulated significantly the rate of [3H]thymidine incorporation into DNA of FSC even during short-term exposures of FSC with KC medium. The growth-promoting effect of normal KC-conditioned medium could be enhanced if the Kupffer cells were challenged with zymosan and phorbol esters, respectively, but not with lipopolysaccharide. The light microscopic appearance of FSC grown in the presence of KC-conditioned media was greatly changed: the cultures became more dense; the cells spread and developed long extensions. The size and number of lipid droplets decreased more rapidly than in control cultures maintained without addition of KC-conditioned media. The protein DNA ratio of FSC exposed with KC-conditioned media was reduced due to a strong increase in DNA, which is not followed by a similar increase in cellular protein. If referred to the DNA content of the culture, the incorporation of [3H]valine into the protein of the medium was not changed, that into cellular protein was strongly reduced under the influence of normal KC-conditioned medium. Secretions of Kupffer cells obviously stimulate significantly the proliferation of FSC in culture.