A semiautomated colorimetric assay (MTT assay), based on the ability of live cells to reduce a tetrazolium-based compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), to a purplish colored formazan product that can be measured spectrophotometrically, has recently been adapted for use in drug sensitivity analysis of cultured human tumor cell lines. We report the application of this assay for the evaluation of the growth factor requirements of human small cell lung cancer (SCLC) cell lines. Specifically, the growth stimulation of each constituent of a previously reported serum-free defined medium system for SCLC including various concentrations of hydrocortisone, insulin, transferrin, 17 beta-estradiol, and selenium (HITES) was evaluated. The optimal concentrations for insulin, transferrin, and selenium derived in the previously reported experiments with direct counting of viable cells were similar to optimal concentrations determined for the growth of three SCLC cell lines (NCI-H82, NCI-N417, NCI-H526) using the MTT assay. In contrast to the previous report, the growth-stimulating effects of hydrocortisone and 17 beta-estradiol were negligible. Using the MTT we have shown that a SCLC cell line, NCI-H345 (which has been previously reported to produce a transferrin-like molecule), was growth-inhibited by an anti-transferrin receptor antibody, when grown in transferrin-free media. The conditioned media from this cell line is stimulatory to other transferrin-sensitive cell lines, suggesting the possibility of an autocrine role for this transferrin-like molecule at least in that cell line. With carefully defined conditions for a given cell line in which cell density and other parameters are within a range of constant MTT metabolism, the assay is well suited for precise analysis of growth factor effects.