The specificity and mode of action of a growth inhibitory factor (GIF) isolated from human plasma-derived serum (PDS) was examined with cell lines established from malignant and nonmalignant human tissues. The mammary cell line, MCF-7, was used in previous work to monitor purification of GIF from serum. The current study showed that, of 9 mammary cell lines, 5 (MDA-MB-415, BT-474, MCF-7, T47D, ZR-75) were inhibited by GIF partially purified from a single serum source. The degree of cell line sensitivity to DEAE-purified GIF was directly related to the amount of inhibition observed with unfractionated PDS. The growth of cells established from other malignancies (lung, colon, melanoma, cervix) and normal diploid fibroblasts was not inhibited. MCF-7 cell growth inhibition was fully reversible following 3 days incubation in GIF but was not reversible after 5 days. Inhibition represented a cytostatic effect. Among the macromolecular synthetic events assayed, DNA and RNA remained unaffected by GIF whereas protein synthesis per cell was markedly elevated. Fluorescence-activated cell sorter analysis of treated and control populations showed no differences in G1, S-, and G2 phase distributions.