Excessive extracellular matrix (ECM) production during dermal wound healing often leads to fibrotic conditions such as keloids and hypertrophic scarring (HSc). Type I collagen is the predominant form of collagen in the human skin and is produced mainly by dermal fibroblasts. It has been suggested that abnormalities in epidermal-dermal interaction can lead to excessive production of collagen by fibroblasts. To identify and further characterize any possible keratinocyte-derived collagen-inhibitory factors (KD-CIFs), we investigated the expression of pro-alpha1(I) collagen at the level of mRNA and protein in human fibroblasts that had been either co-cultured with keratinocytes or treated with keratinocyte-conditioned medium (KCM). Fibroblasts in both groups demonstrated a significant reduction in the steady-state levels of collagen mRNA and protein. Further characterization of KD-CIFs revealed a high-molecular-weight factor (>30 kDa) that showed stable activity at high temperature (56 degrees C) and acidic pH (pH 2). Keratinocyte differentiation did not alter the release of KD-CIFs into KCM. These results provide further evidence that type I collagen expression and synthesis in fibroblasts are regulated by a keratinocyte-releasable factor(s) with an apparent molecular weight between 30 and 50 kDa.