AbstractHuman oocytes were stored (25°C) in 1.5 M MgCl2for 6–30 days, then utilized in the new hemizona assay (HZA) for tight binding of human spermatozoa [Burkman et al.: Fertil Steril 49:688–697, 1988]. We have compared 1) the ability of matching salt‐treated hemizonae or dimethylsulfoxide (DMSO)‐treated hemizonae to distinguish between sperm from semen having normal versus subnormal characteristics and, 2) the kinetics of fertile sperm binding to salt‐treated or DMSO‐treated hemizonae. After sperm preparation, one salt‐treated hemizona was incubated with normal spermatozoa and the matching hemizona was placed with sperm from the subnormal group. As a control, DMSO‐treated hemizonae were incubated in additional sperm droplets. After 4 hours, the number of sperm tightly bound to each hemizona was counted. Within the normal semen group, there was equivalent binding to salt‐ or DMSO‐treated hemizonae (54.0 ± 12 and 49 ± 14, respectively, mean ± SEM). Similarly, tight binding of sperm from the subnormal group was not affected by the zona storage method (21 ± 8 and 17 ± 5, respectively). For either storage approach, binding of subnormal sperm was significantly less (P<0.01) compared with the number of normal sperm attached to the matching hemizona. For the kinetics study, the hemizona binding of proven fertile spermatozoa was followed throughout 8.5 hours. The shape of the binding curve was the same for zonae stored by either method and was consistent with our published kinetics data. Salt storage offers a simple and inexpensive means for accumulating and transporting human zonae pellucida; the resulting hemizonae function effectively in the HZA for estimatin