Cell differentiation in the rat sublingual gland occurs rapidly and is largely complete by birth. To study differentiation of the serous and mucous cells of the sublingual gland, we used antibodies to the secretory proteins CSP-1, SMGB, PSP, and SMGD, and sublingual mucin as specific cell markers. Glands from rats at ages 18, 19, and 20 days in utero, and postnatal days 0, 1, 5, 9, 14, 18, 25, 40, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling. At age 18 days in utero, a few cells in the developing terminal bulbs contained mucous-like apical granules that labeled with anti-mucin. Other cells had mixed granules with a peripheral lucent region and a dense core of variable size that occasionally labeled with anti-SMGD. Additionally, presumptive serous cells with small dense granules that contained CSP-1 and SMGB were present. At age 19 days in utero, the dense granules of these cells also labeled with anti-SMGD. By age 20 days in utero, mucous cells were filled with large, pale granules that labeled with anti-mucin, and serous cells had numerous dense granules containing CSP-1, SMGB, PSP, and SMGD. Fewer cells with mixed granules were seen, but dense regions present in some mucous granules (MGs) labeled with anti-SMGD. After birth, fewer MGs had dense regions, and serous cells were organized into well-formed demilunes. Except for PSP, which was undetectable after the fifth postnatal day, the pattern of immunoreactivity observed in glands of neonatal and adult animals was similar to that seen by age 20 days in utero. These results suggest that mucous and serous cells have separate developmental origins, mucous cells differentiate earlier than serous cells, and cells with mixed granules may become mucous cells. Anat Rec 266:30–42, 2002. © 2002 Wiley-Liss, Inc.