Previous literature suggests that spawning in bivalves is chemically regulated, both by environmental chemical cues and by internal chemical mediators. In a model proposed for zebra mussels, chemicals from phytoplankton initially trigger spawning, and chemicals associated with gametes provide further stimulus for spawning. The response to environmental chemicals is internally mediated by a pathway utilizing serotonin (5-hydroxytryptamine, a neurotransmitter), which acts directly on both male and female gonads. The role of serotonin and most other aspects of the model have been tested only on bivalves other than zebra mussels. The effect of serotonin on zebra mussel spawning was tested. Serotonin (10-5 and 10-3 M) injected into ripe males induced spawning, but injection of serotonin into females did not. Gametes were not released by 10-6 serotonin; in most cases, serotonin injection did not release gametes from immature recipients. Serotonin injection provides a reliable means for identifying ripe male zebra mussels and for obtaining zebra mussel sperm without the need for dissection.