Neurons immunoreactive for the enzyme adenosine deaminase (ADA) in the posterior basal hypothalamus of the rat have a distribution pattern similar to those immunoreactive for histidine decarboxylase (HDC) and are particularly numerous in the tuberal (TM), caudal (CM) and postmammillary caudal (PCM) hypothalamic magnocellular nuclei which harbor neurons containing glutamic acid decarboxylase (GAD). The extent to which these enzymes coexist within neurons of these hypothalamic regions was examined using either serial sections or simultaneous immunostaining for ADA and HDC or GAD in the same section. Analysis of serial sections revealed neuronal coexistence of ADA with HDC or GAD in both TM and CM. In addition some neurons in CM, the only area examined for triple coexistence, were found to contain all three enzymes. In sections processed for ADA simultaneously with HDC or GAD, nearly all ADA-immunoreactive neurons in TM, CM, and PCM as well as those scattered between these nuclei were found to contain HDC, and nearly all contained GAD. Exceptions to this, however, were small cells located lateral to the posterior arcuate nucleus, which appeared to contain ADA but not HDC, and large neurons located at the anterior extreme of TM, which appeared to contain ADA but not GAD. The relatively few ADA- compared with GAD-containing neural systems in brain, together with the presence of ADA in GAD-containing hypothalamic magnocellular neurons, which appear to have widespread projections throughout the brain, indicate that ADA may be a convenient immunohistochemical marker for anatomical investigations of these projections.(ABSTRACT TRUNCATED AT 250 WORDS)