Indirect immunocytochemistry of corticotropin-releasing factor (CRF) and peptide histidine isoleucine amide (PHI) was performed by the use of antibodies raised to CRF and PHI. The staining intensity was quantitated by using an automated microfluorimeter. CRF and PHI immunoreactive fibres showed a similar pattern of distribution in the zona externa of the median eminence of the rat hypothalamus. Administration of colchicine (50 µg i.c.v.) resulted in the appearance of PHI and CRF immunoreactive cell bodies in the parvocellular part of the paraventricular nucleus. The PHI immunoreactive cell bodies were of low intensity and less abundant than those stained with the CRF antisera. Microfluorimetric measurements of the immunostaining in the median eminence showed parallel changes of PHI and CRF immunostaining after adrenalectomy, administration of reserpine and/or pargyline. In order to evaluate whether these data demonstrate that PHI and CRF are colocalized in hypothalamic neurons, we studied the specificity of PHI immunostaining by the use of a nonbiological gelatin model. Although CRF and PHI do not show structural homologies, the PHI antisera caused staining of PHI containing gels (range: 0.001–1 µ M) but also of rat CRF (rCRF)-containing gels (range: 10–300 µ M). In addition, preincubation of one of the PHI antisera with PHI or rCRF both caused a concentration-dependent quenching of the immunostaining in PHI- and CRF-containing gels and in preparations of the median eminence. Again, higher concentrations of rCRF (100 µ M) than PHI (0.1 µ M) were needed to show immunoinhibition, suggesting that the PHI antiserum has much lower avidity for native and fixed rCRF than for native and fixed PHI. We conclude that PHI immunostaining in the rat median eminence as found under the conditions used, is due, at least partially, to cross-reaction of the PHI antisera with rCRF.