In this first report of an effect of caloric restriction on in vivo DNA binding by a chemical carcinogen in rats, hepatic nuclear binding by aflatoxin B1 (AFB) (pmol/mg DNA) in ad libitum-fed (AL) animals was 2.1 times greater than in rats restricted to 60% of AL consumption for 6 weeks. Data indicating more rapid plasma clearance, increased urinary excretion of the toxin, and less microsome-mediated epoxidation of AFB by the restricted group suggest that decreased macromolecular binding may be attributable in part to metabolic alterations. Moreover, various levels of dietary restriction, initiated at different ages, significantly inhibited hepatic DNA synthesis, thus indicating that effects on cell proliferation could also be involved mechanistically. Finally, circulating levels of the lysosomal enzyme, beta-glucuronidase (beta G), were significantly reduced in the restricted rats, and the implications of this finding regarding potential relationships to aging and carcinogenesis are discussed.