Tear lipocalin (TL) (approximately 18 kDa), a member of the lipocalin superfamily, has been identified as one of the major proteins present in rabbit lacrimal fluid. The concentration of TL has been found to be decreased in the tears of patients with dry eye disease. Lacrimal gland insufficiency, one of the major causes of dry eye disease, is known to affect mainly postmenopausal women, where there is a significant decrease in the production of androgen and estrogen. These observations suggest that sex hormones might influence dry eye indirectly by regulating the expression of TL. The purpose of this study was to determine: (1) the effect of sexual maturation on the expression of TL; and (2) if the expression of TL is regulated by the estrogen, 17beta-estradiol, and/or the androgen, dihydrotestosterone, in sexually mature female rabbits. Lacrimal fluid (LF) and lacrimal gland soluble fraction (Si) was collected from juvenile (2 kg) and sexually mature (4 kg) male and female New Zealand white (NZW) rabbits. In addition, LF and Si were collected from 4 kg rabbits, 7 days after being either sham operated (control), ovariectomized (OVX), ovariectomized treated with estrogen (OVX+E) or ovariectomized treated with dihydrotestosterone (OVX+DHT). Samples were analyzed for protein levels of TL by SDS-PAGE and Western blotting using a polyclonal rat anti-rabbit TL antibody. Densitometry analysis showed that TL protein levels in both LF and Si increased with age in male and female rabbits. In addition, TL protein levels were significantly higher in the sexually mature 4 kg male compared with the 4 kg female, while no significant difference in TL protein levels were seen among the juvenile male and female rabbits. Furthermore, ovariectomy decreased the protein levels of TL in LF and Si fraction by 50% and 20% respectively, compared with control values. Estrogen treatment increased TL protein levels by 30% and 50% in the LF and Si fraction respectively, compared with the sham operated group. DHT treatment also increased TL protein levels by approximately 150% in both LF and Si fraction compared with control values. These results support the hypothesis that sex hormones influence TL protein levels in rabbit lacrimal glands. The possibility of a role of TL in dry eye needs to be further investigated.