Female Fischer rats were pair-fed on diets containing either safflower oil (SO) or fish oil (FO) for 6 weeks. Implanted breast 13762 MAT tumors had a doubling times of 35.4 and 55.5 h in SO and FO rats, respectively (P < 0.001). Proliferation kinetics were measured in vivo by bromedeoxyuridine (BrdUrd) labeling and bivariate DNA/BrdUrd analysis by flow cytometry. After 1 h of pulsing, the labeling index was similar in both groups. However, 6 h later, tumor cells from FO rats had significantly lower relative movement of BrdUrd-labeled cells (0.78 vs. 0.91, P < 0.001). These results reflected a significantly longer S phase duration (15.0 vs. 9.1 h, P < 0.001) in FO rats and accounted for all the difference in tumor growth rates. This mechanism, which has not previously been reported, implies a significant role for fatty acids in DNA replication.