Two groups of 10 prepubertal Holstein heifers (86 kg initial body weight) and two groups of 10 postpubertal heifers (292 kg initial body weight) each were maintained under 8 h light: 16 h dark or 16 light: 8 dark photoperiods for an average of 139 days. Then animals were slaughtered, and the total right half of the mammary gland was removed. Mammary parenchymal tissue was separated from surrounding adipose and connective tissue (extraparenchymal tissue). There was no effect of photoperiod on total weight of the mammary gland (parenchymal plus extraparenchymal portions). In contrast, mammary parenchymal weight increased 40 and 30% in prepubertal and post-pubertal heifers exposed to 16 light: 8 dark as compared with heifers exposed to 8 light: 16 dark. Similarly, mammary parenchymal tissue of heifers given 16 light: 8 dark contained 68 and 35% more total deoxyribonucleic acid (an index of cell numbers) than heifers exposed to 8 light: 16 dark. However, photoperiod did not influence concentrations of deoxyribonucleic acid or fat in parenchymal tissue. Extraparenchymal tissue weight was 12 to 35% less in heifers given 16 light: 8 dark than in heifers exposed to 8 light: 16 dark. We conclude that 16 light: 8 dark stimulates the mammary parenchymal tissue to grow into the fat pad (extraparenchymal tissue) of prepubertal and postpubertal Holstein heifers.