Conjugated linoleic acid (CLA) has been reported to have significant activity in inhibiting mammary carcinogenesis. A major objective of this study was to evaluate how changes in the concentration of CLA in mammary tissue as a function of CLA exposure/withdrawal were correlated with the rate of occurrence of mammary carcinomas. Rats treated with a single dose of dimethylbenz[a]anthracene (DMBA) at 50 days of age were given 1% CLA in the diet for either 4 weeks, 8 weeks or continuously following carcinogen administration. No cancer protection was evident in the 4 or 8 week-CLA treatment groups. Significant tumor inhibition was observed only in rats that were given CLA for the entire duration of the experiment (20 weeks). Analysis of CLA in the mammary gland showed that the incorporation of CLA was much higher in neutral lipids than in phospholipids. When CLA was removed from the diet, neutral lipid- and phospholipid-CLA returned to basal values in about 4 and 8 weeks, respectively. The rate of disappearance of neutral lipid-CLA (rather than phospholipid-CLA) subsequent to CLA withdrawal paralleled more closely the rate of occurrence of new tumors in the target tissue. It appears that neutral lipid-CLA may be a more sensitive marker of tumor protection than phospholipid-CLA. However, the physiological relevance of CLA accumulation in mammary lipids is unclear and remains to be determined. A secondary goal of this study was to investigate whether CLA might selectively inhibit clonal expansion of DMBA-initiated mammary epithelial cells with wild-type versus codon 61 mutated Ha-ras genes. Approximately 16% of carcinomas in the control group (without CLA) were found to express codon 61 ras mutation. Although continuous treatment with CLA reduced the total number of carcinomas by 70%, it did not alter the proportion of ras mutant versus wild-type carcinomas, suggesting that CLA inhibits mammary carcinogenesis irrespective of the presence or absence of the ras mutation.