Secretion of a unique collagen by spontaneously transformed murine keratinocytes (PAM cells) in vitro. A spontaneously transformed murine keratinocyte cell line (PAM cell) was found to secrete two nondisulfide-linked collagenous polypeptides with apparent molecular weight (MW) 190-kd and 120-kd. Pulse-chase experiments indicated that the 190-kd polypeptide was secreted into the culture medium in 2 h and processed to the 120-kd collagen component within 4 h. This process was inhibited by EDTA. The 120-kd polypeptide was sensitive to pepsin, and a 50-kd fragment was produced by a mild pepsin treatment at 4 degrees C. A cyanogen bromide peptide map of the 120-kd polypeptide was distinct from that of types I, II, III, IV, and V collagens. These properties indicate similarities to the type VIII-related collagen produced by human astrocytoma cells. The secretion of the collagen rapidly reached a maximum level on the first day of culture and subsequently declined with cell proliferation. An accelerated processing to the 120-kd polypeptide was observed under culture conditions of high cell density. Similar collagens were also found to be produced by normal human keratinocytes. These results indicate that the 120-kd polypeptide is a potentially functional protein that may participate in the formation of the extracellular matrix of keratinocytes. 