A plasma steroid binding protein (SHBP) with medium-high affinity and limited capacity was characterized in the viviparous water snake, Nerodia. This SHBP shows similarity to SHBPs previously described in some other nonmammalian species. A single binding component was detected by Scatchard analyses with a medium-high affinity for testosterone (T), estradiol (E), progesterone (P), and corticosterone (B). Equilibrium dissociation constants ( K d ) for these steroids are as follows: T, 3.6 × 10 −8, M; E, 3.7 × 10 −8, M; P, 5.9 × 10 −8, M; B, 12.1 × 10 −8, M. In competition studies (at saturation) the relative binding affinities (RBA) for E (1.0) and T (1.0) were higher than those for P (0.8) and B (0.59). Further analysis of binding specificity for [ 14H]estradiol at 100-fold excess competitor concentrations revealed that dihydrotestosterone also competes; however, estrone and estriol were relatively poor competitors. Displacement of 3HE by antiestrogen clomiphene derivatives and synthetic estrogen varied: enclomiphene citrate (67.8%), clomiphene citrate (42.2%), diethylstilbestrol (37.3%), and zuclomiphene citrate (15.2%). The SHBP has a relatively high binding capacity ( B max = 0.09−0.7 M), which may be correlated with the relatively high circulating plasma steriod levels in this species. Scatchard analysis, disc gel electrophoresis, sucrose gradient centrifugation, and competition studies indicate the presence of a single moiety binding estradiol, testosterone, progesterone, and corticosterone. The estradiol-SHBP complex is unstable, exhibiting very short times of association ( t < 1.5 min) and dissociation ( K d = 0.0165 sec −1, t 1 2 = 18.3 sec ). Measurement of SHBP levels throughout the seasonal reproductive cycle revealed high levels of binding in previtellogenic, vitellogenic, early pregnant, and postpartum animals. A significantly lower level of SHBP was detected in mid-late pregnancy.