We report the continuous growth maintenance of untransformed and chemically transformed fibroblasts (AKR-2B, AKR-MCA cells) in low concentrations of serum (0.1% FBS). The cell lines established (AKR-0.1F, MCA-0.1F) proliferated at rates comparable to cells maintained under high serum conditions (10% FBS). Complete removal of serum from the cells did not induce quiescence. The MCA-0.1F cells were more similar to the untransformed AKR-2B fibroblasts in their morphology, saturation density, inability to form colonies under anchorage-independent conditions, steady-state level of c-myc expression, and kinetics of induction of c-myc in response to specific growth factors. This report demonstrates the utility of this cell line as a nonquiescent model system for investigating growth factor-specific effects in serum-free, cycling cells. Addition of transforming growth factor-beta (TGF-beta) (5 ng/ml) to proliferating MCA-0.1F cells, in the absence of any serum, induced a multilayered growth pattern at confluency, similar to that of AKR-MCA cells maintained in 10% FBS. Other growth factors tested did not elicit this effect. The induction of this growth pattern by TGF-beta was associated with a sustained induction of the c-myc proto-oncogene at confluency, but not with a restoration of anchorage-independent growth. The data suggest that TGF-beta may play a role in the up-regulation of c-myc at confluency previously described for AKR-MCA cells maintained in 10% serum.