For approximately 10 h before ovulation, female goldfish release the gonadal steroid 17-alpha,20-beta-dihydroxy-4-pregnen-one (17,20-beta-P) to the water where it functions as a potent sex pheromone that induces in males a rapid (15-min) increase in blood gonadotropin (GtH) and a subsequent (4-6 h) increase in milt (sperm and seminal fluid) volume. To determine which olfactory pathways mediate responses to pheromonal 17,20-beta-P, this study examined the effects of various olfactory tract (OT) lesions on the 17,20-beta-P-induced GtH and milt responses in male goldfish. Males remained intact (INTACT) or received either a sham operation (SHAM), complete bilateral OT section (OTX), or bilateral lesions to the lateral (LOTX) or medial (MOTX) subdivisions of the paired OTs. At both 2 days and 1 week after surgery, one-half of each treatment group was exposed to either ethanol (control) or water-bourne 17,20-beta-P for 1 h and then blood sampled for GtH determination. Milt was stripped and collected from all fish 7-8 h after the exposures had commenced. Exposure to water-borne 17,20-beta-P induced increases in GtH and milt volume in INTACT, SHAM, and LOTX animals but failed to evoke these responses in OTX and MOTX fish. The results indicate that central projections of the medial olfactory tracts constitute a functional neuroanatomical pathway by which pheromonal 17,20-beta-P stimulates GtH and milt production in goldfish. In addition, functional and anatomical similarities between this pathway and the mammalian vomeronasal system are discussed.