Immunohistochemistry and image analysis were performed on sections from excessive dermal scar formation to investigate the potential of pericytes to differentiate into collagen-producing cells. Expression of the prolyl-4-hydroxylase beta-subunit (P-4-H) was used as a marker for collagen synthesis as the distribution of this protein was identical to the distribution of procollagen type I C-propeptide and similar to the distribution of cells expressing pro alpha 1(I) collagen mRNA. Double immunofluorescence stainings using combinations of monoclonal antibodies specific for activated pericytes in vivo (high molecular weight-melanoma associated antigen (HMW-MAA)), P-4-H, smooth muscle alpha-actin (SMA), endothelial cells (PAL-E), platelet-derived growth factor (PDGF) beta-receptor, and the integrin alpha 5 subunit were performed. Stained sections were analyzed by computerized image analysis allowing for a quantification of the degree of colocalization between pairs of antigens on the same tissue section. Four different subpopulations of HMW-MAA expressing cells were discerned. The first subpopulation corresponded to intramural pericytes, juxtapositioned to the endothelium, that expressed HMW-MAA, SMA, integrin alpha 5 subunit and the PDGF beta-receptor, but not P-4-H. The second subpopulation was partly dissociated from the microvascular wall and exhibited a similar antigen expression except for a decrease in expression of SMA. Cells in the third subpopulation were located in the perivascular space and expressed P-4-H, integrin alpha 5 subunit, the PDGF beta-receptor and, albeit less pronounced, HMW-MAA, but not SMA. The fourth subpopulation expressed integrin alpha 5 subunit, HMW-MAA and the PDGF beta-receptor, no expression of SMA and a strong expression of P-4-H. Moreover, an in vitro analysis of cells derived from isolated microvascular fragments from human dermis revealed a similar pattern of phenotypical change. Taken together the data suggest that a population of intramural pericytes migrate into the perivascular space and develop into collagen-synthesizing fibroblasts during fibrosis.