Fibroblast heterogeneity in human periodontal tissues was characterized by cloning and immuno-histochemical techniques. Cell suspensions from primary cultures gingival (GF) and PDL fibroblasts (PF) were cloned. The relative intensity of double-labeled immunofluorescence, using specific antibodies to the extracellular matrix (ECM) molecules collagen type I (CI), type III (CIII), and fibronectin (Fn), was measured by photometry. Most clones derived from either GF or PF showed positive intracellular staining for both CI and Fn, and CIII and Fn. However, there were variations in fluorescence intensity for CI and Fn, ranging from relatively weak to strongly positive. The fluorescence for CI and CIII was relatively weak in most isolated GF clones in contrast to their PF clones. These observations coupled with studies of growth and cellular morphology in individual clones suggest that: 1) GF and PF contain functionally heterogeneous subpopulations; and 2) the synthesis and expression of extracellular matrix molecules of GF may be essentially different from that of PF.