The purpose of this study was to examine whether human gingival fibroblasts produce a cytokine which modulates in immune and inflammatory responses including alterations in connective tissue metabolism in periodontal tissue. We found that a cultured human gingival fibroblast cell line (Gin-1) and freshly isolated human gingival fibroblasts produced thymocyte-activating factor(s), so we called the factor(s) fibroblast-derived thymocyte-activating factor (FTAF). Growth of the producing cell was itself modulated by the factor(s). Gin-1 cells spontaneously produced a significant amount of FTAF in a cell growth-dependent manner. Maximum activity was observed in conditioned medium from stationary-phase cells. The activity in conditioned medium of cultures lacking serum was significantly higher than that in those containing serum. Treatment of Gin-1 cell cultures with cycloheximide, an inhibitor of protein synthesis, markedly inhibited FTAF production. When Gin-1 cells were stimulated by triggering with muramyl dipeptide or sonicated extracts of Bacteroides gingivalis, FTAF production was significantly stimulated. Freshly isolated human gingival fibroblasts from gingival biopsies of healthy donors also produced FTAF which enhanced thymocyte proliferation. Peaks of thymocyte proliferation activity in conditioned medium from Gin-1 cells were observed in fractions having molecular weights of 25,000, 35,000, and 45,000, as determined by Sephadex G-75 column chromatography. The peak fractions (partially purified FTAF) significantly suppressed the proliferation of Gin-1 cells themselves as evaluated by [3H]thymidine uptake. The suppressive effect of partially purified FTAF was, at least partially, mediated by endogenous prostaglandin for the following reasons: addition of indomethacin, and inhibitor of prostaglandin synthesis, abrogated the suppressive effect; partially purified FTAF stimulated the production of prostaglandin E2 by the cells; and the suppression of cell proliferation was reinforced by addition of exogenous prostaglandins. These observations suggest that gingival fibroblasts play a significant role in regulation of cell growth of lymphocytes and in their own growth under physiological conditions and in pathological states in periodontal connective tissue.