We have produced a set of bacterial expression proteins corresponding to 10 segments of tenascin and two of fibronectin and tested them for heparin binding and cell adhesion. We used polymerase chain reaction cloning to terminate the segments precisely at domain boundaries. Heparin binding activity was mapped to two different tenascin segments: one comprising the fourth and fifth fibronectin type III domains, and to TNfbg, the fibrinogen-like terminal knob. TNfbg, but none of the other tanascin segments, also supported adhesion of primary rat embryo skin fibroblasts. The fibroblasts did not spread on TNfbg but remained rounded. Cell binding to TNfbg occurred in the presence or absence of divalent cations and was not inhibited by RGD peptides, suggesting that integrins are not involved. Fibroblast binding to TNfbg was strongly inhibited by soluble heparin, by treating the cells with heparitinase, or by culture conditions that cause undersulfation of proteoglycans. These observations suggest that cell attachment to TNfbg is mediated by cell surface proteoglycans. We have also made full-length cDNA constructs for the largest and smallest splice variants of human tenascin, as well as one truncated after the 14th epidermal growth factor-like domain, in the pNUT mammalian cell expression vector. Stably transfected baby hamster kidney cell lines secreted large quantities of tenascin, and this was assembled into normal hexabrachions, the arm length corresponding to the construct.