Serotonin (5-HT) is a potent regulator of adult neurogenesis in the crustacean brain, as in the vertebrate brain. However, there are relatively few data regarding the mechanisms of serotonin's action and which precursor cells are targeted. Therefore, we exploited the spatial separation of the neuronal precursor lineage that generates adult-born neurons in the crayfish (Procambarus clarkii) brain to determine which generation(s) is influenced by serotonin, and to identify and localize serotonin receptor subtypes underlying these effects. RT-PCR shows that mRNAs of serotonin receptors homologous to mammalian subtypes 1A and 2B are expressed in P. clarkii brain (referred to here as 5-HT1α and 5-HT2β). In situ hybridization with antisense riboprobes reveals strong expression of these mRNAs in several brain regions, including cell clusters 9 and 10 where adult-born neurons reside. Antibodies generated against the crustacean forms of these receptors do not bind to the primary neuronal precursors (stem cells) in the neurogenic niche or their daughters as they migrate, but do label these second-generation precursors as they approach the proliferation zones of cell clusters 9 and 10. Like serotonin, administration of the P. clarkii 5-HT1α-specific agonist quipazine maleate salt (QMS) increases the number of bromodeoxyuridine (BrdU)-labeled cells in cluster 10; the P. clarkii 5-HT2β-specific antagonist methiothepin mesylate salt (MMS) suppresses neurogenesis in this region. However, serotonin, QMS and MMS do not alter the rate of BrdU incorporation into niche precursors or their migratory daughters. Our results demonstrate that the influences of serotonin on adult neurogenesis in the crayfish brain are confined to the late second-generation precursors and their descendants. Further, the distribution of 5-HT1α and 5-HT2β mRNAs and proteins indicate that these serotonergic effects are exerted directly on specific generations of neuronal precursors. Taken together, these results suggest that the influence of serotonin on adult neurogenesis in the crustacean brain is lineage dependent, and that 5-HT1α and 5-HT2β receptors underlie these effects.