In addition to its anti-coagulant effect, heparin inhibits the growth of several types of cells. Recent studies suggest that heparin inhibition of proliferation of cultured human keratinocytes, results primarily from interaction with keratinocyte-generated, heparin-binding autocrine growth factors. In this study, we evaluated whether non-anticoagulant heparin analogs, and oligosaccharide fragments of heparin, retain the growth-inhibitory properties of whole heparin on human keratinocytes. Second-passage neonatal keratinocytes were grown in serum-free keratinocyte growth medium, and the effect of heparin analogs was studied in the absence of exogenous growth factors using keratinocyte-conditioned medium. Cell proliferation was assessed by measurement of both DNA content and [3H]-thymidine incorporation. The addition of heparin inhibited the conditioned medium-stimulated keratinocyte proliferation in a dose-dependent manner, with 80% inhibition at or above 10 micrograms/ml. Moreover, heparin was not toxic to keratinocytes (as detected by propidium-iodide fluorescence and by retention of normal protein synthetic rate) and it did not induce terminal differentiation (as measured by cornified envelope formation). Furthermore, heparin stimulated protein secretion by keratinocytes without altering rates of protein synthesis. The growth-inhibitory effects of heparin oligosaccharides were directly proportional to their chain length. The hexasaccharide unit represented the minimum requirement for inhibition, whereas decasaccharide units demonstrated nearly equivalent growth inhibition to native heparin. Finally, two non-anticoagulant heparin analogs were equipotent with heparin in inhibiting autocrine-induced keratinocyte growth. These studies show that the growth-inhibitory activities of heparin are independent of the anticoagulant effects and that decasaccharides contain the optimal oligosaccharide chain length for the antiproliferative effect in human keratinocytes.