Tendons from 14--17-day-old chick embryos contain predominantly type I collagen and about 5% AB2 collagen; type III collagen is not detectable by biochemical methods, such as sodium dodecyl sulfate/polyacrylamide gel electrophoresis or cyanogen bromide pattern, but can be visualized by immunofluorescence staining with collagen-type-specific antibodies. Similarly, freshly dissociated tendon cells secrete only type I collagen into the culture medium but no significant amounts of type III collagen [Uitto, J., Lichtenstein, J. R., and Bauer, E. A. (1976) Biochemistry, 15, 4935--4942]. Transfer of tendon cells from chick embryos to monolayer conditions, however, initiated synthesis of type III collagen in about 10% of the cells within three days, as visualized by immunofluorescence staining. Secretion of type III collagen into the culture medium can also be detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. With increasing number of passages the number of cells producing type III collagen reached levels of about 80% after the third passage, while 90% of all cells stained positively for type I collagen. This is reflected by an increase of production of type III collagen as determined by CM-cellulose chromatography. Using velocity sedimentation, the secretion of type III procollagen and of pN-collagen (carrying the amino-terminal extension only), into the culture medium of a second-passage tendon cell culture was detected. This study provides new evidence that the phenotype of cells may alter during transfer from the environment in vivo to conditions in vitro and that additional changes may occur with time in culture.