The density of vasopressin-immunoreactive (AVP-ir) fibers in the lateral septum and lateral habenular nucleus is lower in prairie vole fathers--which display paternal behavior under natural conditions-than in sexually naive males. To see if these changes occur before or after the birth of pups, and whether they are related to changes in paternal behavior, we tested paternal responsiveness and measured AVP-ir fiber density in the lateral septum, lateral habenular nucleus, medial preoptic area, and paraventricular nucleus of the thalamus of sexually naive males and females (0P) and breeding pairs that were sacrificed shortly after mating (3P); during early (13P); or late gestation (21P); or after the birth of pups (6PP). Paternal responsiveness was increased in 3P males and reached a plateau in 13P males. AVP-ir fiber density did not change in the medial preoptic area and the paraventricular nucleus of the thalamus. The fiber density in the lateral septum and lateral habenular nucleus was affected differently in males and females. Among males, 3P animals had the lowest fiber density, while 13P and 6PP animals had an intermediate, and 0P and 21P animals the highest fiber density, whereas among females, no differences in fiber density were found. A second experiment showed that the decrease in fiber density in 3P males could be induced by cohabitation with an unfamiliar female but not by an unfamiliar male nor by relocation to a novel cage. The changes in AVP-ir fiber density shortly after mating suggest that these fibers may be involved in paternal responsiveness as well as in various other social behaviors that change after mating.