Growth characteristics and macromolecular synthesis of fibroblasts derived from human periodontal ligament (PDLF) and gingiva (GF) have been compared in cell culture. Cells were isolated from explants and plated at 500,000 cells/100 mm culture dish (day 0) with daily changes of culture medium. DNA histograms were obtained by flow microfluorimetric analysis to confirm the growth state of the cell cultures. Human PDLF cultures became confluent at day 6 as determined by cell number and cell cycle analysis while GF were confluent by day 4. Initially, DNA content of logarithmically growing cells was significantly greater in GF cultures; however, when confluent, DNA content and cell number was greater in PDLF cultures. Total protein content in GF was slightly greater than PDLF until day 7 but this difference was not significant. Analysis of collagen and noncollagen protein synthesis revealed a greater trend in noncollagen protein synthesis in the GF cultures compared to PDLF cultures. Analysis of glycosaminoglycans in the culture medium of GF and PDLF revealed similar distributions of components. In the cellular fraction, GF had greater amounts of hyaluronic acid and heparin and lesser amounts of chondroitin sulfates A and C than PDLF cultures. The results indicate that the growth characteristics of PDLF and GF, although similar in many respects, do exhibit specific differences in proliferative rates and macromolecular synthesis. The differences observed in these parameters may be important during in vivo events, such as guided tissue regeneration, where significant functional differences are observed between gingival connective tissue and periodontal ligament connective tissue.