By using a recently modified method of isolating and culturing rabbit corneal cells, this study investigated the presence of a diffusible substance(s) in stromal fibroblast conditioned medium that stimulated the growth of cultured corneal epithelial cells. The growth stimulation involved initiation of DNA synthesis (assayed by [3H]-thymidine incorporation) and enhanced cell proliferation (quantified by cell counting). Among the three corneal cell types, only fibroblasts (rabbit and human) released the stimulatory substance, which acted only on epithelial cells. The effect of this stromal fibroblast factor (SFF) was observed after an exposure period of less than 16 hr and persisted as long as it was present. Its action was concentration-dependent and was not a result of improvement in the survival of epithelial cells during culture. Both sparse and confluent epithelial cultures were susceptible to SFF. The release of SFF was correlated with the number of fibroblasts in the culture and appeared to be sensitive to the growth condition of the cells. Both the release and action of SFF did not depend on the presence of serum in the culture medium. The factor was heat resistant and insensitive to proteolytic enzymes. From ultrafiltration studies, the size of SFF was estimated to be in the approximate range of 50-1000 daltons. By direct comparison of the stimulatory effect with other previously studied growth promoting agents, it was concluded that SFF was not epidermal growth factor, fibroblast growth factor, putrescine, cyclic AMP, hydrocortisone or acetate. The implication of SFF in the regulation of epithelial growth by endogenous, intercellular mechanisms is discussed.