A combined anterior pituitary (CAP) function test was assessed in eight healthy male beagle dogs. The CAP test consisted of sequential 30-second intravenous administrations of four hypothalamic releasing hormones in the following order and doses: 1 microgram of corticotropin-releasing hormone (CRH)/kg, 1 microgram of growth hormone-releasing hormone (GHRH)/kg, 10 micrograms of gonadotropin-releasing hormone (GnRH)/kg, and 10 micrograms of thyrotropin-releasing hormone (TRH)/kg. Plasma samples were assayed for adrenocorticotropin, cortisol, GH, luteinizing hormone (LH), and prolactin (PRL) at multiple times for 120 min after injection. Each releasing hormone was also administered separately in the same dose to the same eight dogs in order to investigate any interactions between the releasing hormones in the combined function test. Compared with separate administration, the combined administration of these four hypothalamic releasing hormones caused no apparent inhibition or synergism with respect to the responses to CRH, GHRH, and TRH. The combined administration of these four hypothalamic releasing hormones caused a 50% attenuation in LH response compared with the LH response to single GnRH administration. The side effects of the combined test were confined to restlessness and nausea in three dogs, which disappeared within minutes after the administration of the releasing hormones. It is concluded that with the rapid sequential administration of four hypothalamic releasing hormones (CRH, GHRH, GnRH, and TRH), the adenohypophyseal responses are similar to those occurring with the single administration of these secretagogues, with the exception of the LH response, which is lower in the CAP test than after single GnRH administration.