Neuropeptides, including oxytocin-like peptides, are a conserved group of hormones that regulate a wide range of social behaviors, including vocal communication. In the current study, we evaluate whether putative brain sites for the actions of isotocin (IT), the oxytocin (OT) homolog of teleost fishes are associated with vocal courtship and circuitry in the plainfin midshipman fish (Porichthys notatus). During the breeding season, nesting males produce advertisement calls known as "hums" to acoustically court females at night and attract them to nests. We first identify IT receptor (ITR) mRNA in evolutionarily conserved regions of the forebrain preoptic area (POA), anterior hypothalamus (AH), and midbrain periaqueductal gray (PAG), and in two topographically separate populations within the hindbrain vocal pattern generator-durationcoding vocal prepacemaker (VPP) and amplitude-coding vocal motor nuclei (VMN) that also innervate vocal muscles. We also verify that ITR expression overlaps known distribution sites of OT-like immunoreactive fibers. Next, using phosphorylated ribosomal subunit 6 (pS6) as a marker for activated neurons, we demonstrate that ITR-containing neurons in the anterior parvocellular POA, AH, PAG, VPP, and VMN are activated in humming males. Posterior parvocellular and magno/gigantocellular divisions of the POA remain constitutively active in nonhumming males that are also in a reproductive state. Together with prior studies of midshipman fish and other vertebrates, our Abbreviations: ac, anterior commissure; AH, anterior hypothalamus; AT, anterior tuberal nucleus of hypothalamus; Cc, cerebellar crest; Cg, granule cell layer of the corpus of the cerebellum; Cm, molecular layer of the cerebellum; CP, central posterior nucleus; Cpost, posterior commissure; D, area dorsalis of telencephalon; Dc, central zone of D; Dd, dorsal zone of D; DIL, diffuse nucleus of the inferior lobe; DiV, diencephalic ventricle; Dl, lateral zone of D; Dm, medial zone of D; Dm-p, posterior division of Dm; DOdm, dorsomedial division of descending octaval nucleus; DOri/I, rostral intermediate/intermediate division of descending octaval nucleus; DPo, dorsal posterior nucleus of the thalamus; ECL, external cellular layer of the olfactory bulb; GL, glomerular layer of olfactory bulb; Had, dorsal division of the habenula; Hav, ventral division of the habenula; Hb, habenula; Hd, dorsal zone, periventricular hypothalamus; HoCo, horizontal commissure; Hv, ventral zone,