Conjugated linoleic acid (CLA) is a minor fatty acid found predominantly in the form of triglycerides in beef and dairy products. Previous work by Ip and co-workers showed that free fatty acid-CLA at < or = 1% in the diet is protective against mammary carcinogenesis in rats. The present study verified that the anticancer activities of free fatty acid-CLA and triglyceride-CLA are essentially identical. This is an important finding, because it rules out a nonspecific free fatty acid effect. In terms of practical implication, we can continue the in vivo research with the less-expensive free fatty acid-CLA without compromising the physiological relevance of the data. A primary objective of this report was to investigate how the timing and duration of CLA feeding might affect the development of mammary carcinogenesis in the methylnitrosourea (MNU) model. We found that exposure to 1% CLA during the early postweaning and pubertal period only (from 21 to 42 days of age) was sufficient to reduce subsequent tumorigenesis induced by a single dose of MNU given at 56 days of age. This period incidentally corresponds to a time of active morphological development of the mammary gland to the mature state. In contrast to the above observation, a continuous intake of CLA was required for maximal inhibition of tumorigenesis when CLA feeding was started after MNU administration, suggesting that some active metabolite(s) of CLA might be involved in suppressing the process of neoplastic promotion/progression.