The mammillary bodies represent important relay stations for one of the major neuronal circuits in the brain: the limbic circuit. Mammillary projections traveling through the principal mammillary tract are established early during development, forming the mammillotegmental bundle, which appears fully developed by embryonic day 15 (E15). The mammillothalamic tract develops later, around E17-E18, forming a compact system of collateral fibers originating from the principal mammillary tract and reaching the thalamus by E20. The Pax-6 gene is expressed in various regions of the developing brain, among which the border separating the ventral thalamus from the dorsal thalamus, known as the zona limitans intrathalamica, is especially significant. In this report, the development of the efferent mammillary system of fibers was studied in wild type and Pax-6 mutant mice by using carbocyanine tracers and Golgi preparations. In mutant mice, the mammillotegmental bundle developed normally; however, the mammillothalamic tract was missing. By using anti-Pax-6 antibodies in wild type mice, the existence of an immunoreactive cell cluster is described surrounding the bifurcation point of the principal mammillary tract. The results of this study suggest that there is a correlation of these cells with a particular type of Golgi impregnated neuron.