Introduction. In the alpaca diencephalon, the distribution of immunoreactive cell bodies and fibers containing methionine-enkephalin (MET) or substance P (SP) has been studied. Material and methods. The immunohistochemical study was performed by standard method on the dien-cephalon of four male alpacas that lived at sea level. Results. Nerve fibers containing MET or SP were widely distributed in the thalamus and hypothalamus. MET-and SP-immunoreactive fibers showed a similar distribution in the whole diencephalon. Immunoreactive cell bodies containing MET or SP were only observed in the hypothalamus. The distribution of MET-immuno-reactive cell bodies was more widespread than that observed for cell bodies containing SP. Conclusions. A close neuroanatomical relationship between the tachykininergic (SP) and enkephalinergic (MET) systems was observed in the whole diencephalon suggestive of the existence of multiple physiological interactions between both systems. (Folia Histochemica et Cytobiologica 2020, Vol. 58, No. 2, 135–146).