Ovarian factors that promote growth of vascular smooth muscle cells were investigated. Two distinct heparin-binding polypeptides were isolated from bovine follicular fluid by successive chromatographies. N-Terminal and tryptic peptide fragment analysis of these polypeptides revealed that they are identical to midkine (MK) and pleiotrophin (PTN), respectively, which form a new family of heparin-binding growth/differentiation factors. Two N-terminally distinct forms of midkine were also identified in the bovine follicular fluid. The concentrations of MK and PTN in the bovine follicular fluid were estimated to be 125 micrograms/l and 400 micrograms/l, respectively. The present findings that MK and PTN are quite rich in the follicular fluid will provide a new insight into so far unclarified functions of MK and PTN, especially their roles in the maturation of ovarian follicles.