We compared fresh and frozen-thawed cynomolgus monkey spermatozoa tight binding to the zona pellucida under hemizona assay (HZA) conditions. Monkey oocytes were recovered after superovulation and stored in salt solution. Matching hemizonae were obtained by micromanipulation. Semen, obtained by electroejaculation, was used fresh or was cyropreserved, thawed, and washed by swim-up separation. At the standard initial dilution of 500,000 motile sperm/ml (or 5 x 10(4) motile sperm/hemizona), binding was significantly higher for fresh sperm (P = 0.00004). For frozen-thawed samples, there was a linear increase in the number of tightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 x 10(6) motile sperm/hemizona, binding of frozen-thawed spermatozoa was similar to that of fresh at a standard concentration. Kinetic studies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evaluation of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.