Objective This study assessed the effect of varying prenatal protein levels on the development of homing orientation in rat pups. Methods Long-Evans rats were fed one of the four isocaloric diets containing 6% (n = 7 litters), 12% (n = 9), 18% (n = 9), or 25% (n = 10) casein prior to mating and throughout pregnancy. At birth, litters were fostered to well-nourished control mothers fed a 25% casein diet during pregnancy, and an adequate protein diet (25% casein) was provided to weaning. On postnatal days 5, 7, 9, 11, and 13, homing behaviors, including activity levels, rate of successful returns to the nest quadrant and latencies to reach the nest over a 3-minute test period were recorded from two starting positions in the home cage. Adult body and brain weights were obtained at sacrifice (postnatal day 130 or 200). Results Growth was impaired in pups whose mothers were fed a 6% or, to a lesser extent, a 12% casein diet relative to pups whose mothers were fed the 18 and 25% casein diets. The 6 and 12% prenatal protein levels resulted in lower activity levels, with the greatest reduction on postnatal day 13. However, only the 6% pups had reduced success and higher latencies in reaching the nest quadrant when compared with pups from the three other nutrition groups. Latency in reaching the nest quadrant was significantly and negatively associated with adult brain weight. Discussion Home orientation is a sensitive measure of developmental deficits associated with variations in prenatal protein levels, including levels of protein deficiency that do not lead to overt growth failure.