The ability to store turkey semen for 24 h in vitro without a significant loss in fertility would benefit the commercial turkey industry. We investigated the use of exogenous phosphatidylcholine to improve the viability, mobility, hydrolyzing ability, and fertility of stored turkey sperm. For experiment 1, semen was diluted 1:1 with extender containing 0.0 (control), 0.5, 2.5, or 10.0 mg/mL of phosphatidylcholine labeled with a fluorochrome and maintained under aerobic conditions for 24 h at 4°C. Semen aliquots were removed at 30-min intervals during the first 4 h and at 1-h intervals from 8 to 24 h of storage for fluorometric evaluation by flow cytometry. Turkey sperm incorporated labeled phosphatidylcholine in a dose-dependent manner during the first 12 h of storage (P<0.05). At 24 h of storage, phosphatidylcholine uptake increased 7.8-fold for the 0.5-mg treatment, 9.2-fold for the 2.5-mg treatment, and 6.7-fold for the 10-mg labeled phosphatidylcholine treatment. For experiment 2, turkey semen was diluted and stored as for experiment 1 except the phosphatidylcholine was unlabeled. After 24 h, the viability, mobility, hydrolyzing ability, and fertility of turkey sperm was assessed. Supplemental phosphatidylcholine did not improve (P>0.05) the viability or mobility of stored sperm. At 2.5 mg/mL, phosphatidylcholine improved the hydrolyzing ability of stored sperm compared with control or other phosphatidylcholine treatments (P<0.05). The mean fertility rate of eggs from hens inseminated with control semen was 33.5%±4.5. Use of 0.5, 2.5, or 10.0 mg phosphatidylcholine/mL improved (P<0.05) the fertility rates during the first 11 wk of egg production; higher fertility rates occurred with 2.5 mg phosphatidylcholine/mL compared with other phosphatidylcholine treatments for 5 of those 11 wk (P<0.05). We conclude that supplemental phosphatidylcholine appears to counteract the damaging effects of lipid peroxidation or enzymatic degradation during in vitro storage by providing exogenous phospholipids for incorporation into the turkey sperm plasma membrane.