Bird song is controlled by a discrete network of brain nuclei. The size of several song control nuclei changes seasonally in many seasonally breeding songbird species. Reports of seasonal changes in the size of song nuclei have relied primarily on Nissl stains to define the borders of these regions. Recent studies found that the size of the song nucleus higher vocal center (HVC) in male canaries did not change seasonally when its borders were defined by histological markers other than Nissl staining. We used three labels to define the borders of the HVC in male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii): Nissl staining, the distribution of acetylcholinesterase-positive neuropil, and the distribution of neurons projecting to another song nucleus, area X. The HVC was larger in males exposed to a breeding photoperiod and testosterone concentrations than in males exposed to a nonbreeding photoperiod and testosterone concentrations, regardless of which of these three methods was used to define the borders of the HVC. This result suggests that seasonal changes in the Nissl-defined borders of the HVC reflect changes in the distribution of physiologically relevant markers of the nucleus and are not merely artifacts of the Nissl-staining method.