Cryopreservation of sperm is seen as an important step in developing effective hatchery culture techniques for the black-lip pearl oyster, Pinctada margaritifera. As a preliminary investigation into cryopreservation of the gametes of this species we tested 5 cryoprotectant agent combinations for their ability to retain sperm motility: (1) 1 M trehalose and 5, 10 and 15% dimethyl sulphoxide (DMSO); (2) Hanks calcium-free balanced salt solution (C-F HBSS) and 5%, 10% and 15% DMSO; (3) C-F HBSS and 5, 10 and 15% propylene glycol (PG); (4) 1 M trehalose and an equal combination of DMSO and PG making up 5, 10, 15% total volume; and (5) C-F HBSS and an equal combination of DMSO and PG making up 5%, 10% and 15% total volume. Total, rapid and progressive sperm motilities were estimated through computer assisted sperm analysis (CASA). Sperm cryopreserved in 1 M trehalose and 5% DMSO retained the highest total (86.0 ± 1.2% SE), progressive (46.0 ± 1.2% SE) and rapid (25.1 ± 0.6% SE) motilities of all cryoprotectant solutions, whereas those cryopreserved with PG generally retained poor motility. Although the 1 M trehalose and 5% DMSO treatment compared favorably with that of fresh sperm for total motility (P < 0.01), all cryoprotectant treatments were poor at retaining the proportion of original rapid and progressively moving sperm. This study highlights the potential for cryopreservation of gametes from P. margaritifera, which will benefit selective breeding and conservation programs with this important commercial species.