A serum-free, hormone-free medium (SF2) was designed for the Nb2 rat lymphoma bioassay for lactogens as batches of horse serum (HS), which were commonly used, were found to be inconsistent in their suitability and to contain factors modulating the PRL-induced growth response of clone Nb2-11C. In a 3-day incubation with less than 500 pg/ml human GH (hGH), SF2 was better than the traditional medium in supporting Nb2-11C growth, although the comparative efficiency of SF2 decreased at higher hGH levels. Known growth factors (epidermal growth factor, fibroblast growth factor, platelet derived-growth factor, recombinant somatomedin-C, multiplication-stimulating activity) and insulin had no consistent effect on the cell growth in SF2 either in the presence or absence of hGH. Corticosterone (12.4-150 nM) was toxic to the Nb2-11C cells. SF2 could support the growth of Nb2-11C cells for at least 30 passages in the presence of 5 ng/ml hGH, and that of 2 spontaneously proliferating cell lines (Nb2-SP and Nb2-HSP) for the same length of time in the absence of lactogen. However, in all cases the growth rate in SF2 was lower than that seen in the presence of 10% HS. Long-term culture of Nb2-SP and Nb2-HSP cells in SF2 led to an increase of the growth rate with time. There was a change in the responsiveness of Nb2-SP cells to lactogens after long-term culture in SF2 which was only apparent in the presence of HS. After 10 passages in SF2, Nb2-11C cells showed no apparent changes in lactogen-induced growth response, cell phenotype, cell size, or binding capacity for [125I]hGH.