We detected a close morphological association between melanin-concentrating hormone (MCH)-immunoreactive (ir) fibers and somatolactin (SL)-ir cells in the pars intermedia of the cichlid fish Cichlasoma dimerus by double-label immunofluorescence. Male pituitaries obtained from adult C. dimerus were incubated with 0.1–10 μM salmon MCH, and the amount of SL released into the culture medium was semi-quantified by Western blot. This assay showed an increase of SL release in a dose-dependent manner (linear regression: P < 0.05). A close association of GnRH-ir fibers with SL-ir cells was also detected at the pars intermedia level. Male pituitaries were also incubated with 0.1–10 μM of mammalian GnRH, and SL release was semi-quantified by Western blot, showing an increase of released SL levels in a dose-dependent manner (linear regression: P < 0.05). In contrast, SL release was unaffected from female pituitaries incubated with salmon MCH; however, an increasing tendency was observed when mammalian GnRH was used. Hypothalamic close association of MCH-ir perikarya and GnRH-ir fibers was found by double-label immunofluorescence indicating a possible relationship between them. These results suggest that SL, like other pituitary hormones, is under hypothalamic control and is involved in diverse physiological processes including background adaptation and reproduction. This study has also shown that the in vitro culture of a single C. dimerus pituitary is a feasible method for studying the control of SL release and other pituitary hormones.