Binding studies of metallothionein and rat spermatozoa were performed using 125I-Tyr-metallothionein (1251-MT). Reactions between 1251-MT and spermatozoa indicated that MT bound in two forms , namely, middle affinity (Kdl=2 x 10-9 M) binding and low affinity (Kd2=1 x 10_8 M) binding. Labeled MT binding to spermatozoa was inhibited by adding anti-MT antibody. Total binding reactions of MT were temperature and incubation time dependent. By transmission electron microscopy using a gold conjugate (indirect method), gold particles bound to MT were shown to bind to the cell membrane of the head and the proximal portion of the tail. By optical autoradiography, grains of labeled MT were localized mainly in the head and the proximal portion of the tail. By electron microscopical autoradiography, grains of labeled MT were identified mainly in the cell membrane of the head and tail and partly in the nucleus. These results suggest that MT has both specific and non-specific binding sites on the spermatozoal membrane. metallothionein (MT); spermatozoa; autoradiography; transmission electron microscopy Metallothionein (MT) is a metal-binding protein with a low molecular weight of approximately 6,000 and with high cysteine and sulfur content (Margoshes and Vallee 1957). It has been identified in a variety of organs, including the prostate (Waalkes et al. 1982;Bataineh et al. 1986;Umeyama et al. 1987;Suzuki et al. 1991), and binds class II-B metals, such as cadmium and zinc. Zinc is concentrated in the prostatic tissue and secreted into prostatic fluid (Mawson and Fisher