Sex differences in growth hormone (GH) secretion in the rat are thought to be determined, to a large extent, by gonadal steroid‐dependent sex differences in somatostatin (SRIH) secretion from neurones in the periventricular nucleus (PeN) which project to the median eminence (ME). The present study aimed to obtain direct evidence for sex differences and gonadal regulation of SRIH release within this pathway and to determine the relationships between SRIH mRNA expression, SRIH peptide content and SRIH secretion in the adult rat. Somatostatin mRNA expression in the PeN and peptide content in both PeN and ME were higher in males than females (P<0.05). However, both basal and 56 m m K⁺‐stimulated SRIH release in vitro from hypothalamic explants incorporating the PeN–ME pathway were higher (P<0.01) in females. The gonadectomy of female rats resulted in significantly reduced basal levels of SRIH release equivalent to that of males but had no effect on SRIH mRNA/peptide content or K⁺‐stimulated release. In contrast, gonadectomy of male rats reduced SRIH mRNA and peptide contents and elevated K⁺‐stimulated secretion (P<0.01) to levels similar to that seen in intact females, without affecting basal release. In summary, these results demonstrate that in the PeN‐ME of the adult rat: (1) SRIH mRNA and peptide content is well correlated and sexually dimorphic but dependent on gonadal factors in the male only; (2) SRIH secretion is sexually dimorphic and dependent on gonadal factors; but (3) differences in mRNA/peptide content do not reflect secretory capacity; and (4) gonadal factors differentially modulate SRIH secretory dynamics in males and females.