Nonsteroidal anti-inflammatory drugs are widely reported to inhibit carcinogenesis in humans and in rodents. These drugs are believed to act by inhibiting one or both of the known isoforms of cyclooxygenase (COX). However, COX-2, and not COX-1, is the isoform most frequently reported to have a key role in tumor development. Here we report that homozygous deficiency of either COX-1 or COX-2 reduces skin tumorigenesis by 75% in a multistage mouse skin model. Reduced tumorigenesis was observed even though the levels of stable 7,12-dimethylbenz(a)anthracene-DNA adducts were increased about 2-fold in the COX-deficient mice compared with wild-type mice. The premature onset of keratinocyte terminal differentiation appeared to be the cellular event leading to the reduced tumorigenesis because keratin 1 and keratin 10, two keratins that indicate the commitment of keratinocytes to differentiate, were expressed 8-13-fold and 10-20-fold more frequently in epidermal basal cells of the COX-1-deficient and COX-2-deficient mice, respectively, than in wild-type mice. Papillomas on the COX-deficient mice also displayed the premature onset of keratinocyte terminal differentiation. However, loricrin, a late marker of epidermal differentiation, was not significantly altered, suggesting that it was the early stages of keratinocyte differentiation that were primarily affected by COX deficiency. Because keratin 5, a keratin associated with basal cells, was detected differently in papillomas of COX-1-deficient as compared with COX-2-deficient mice, it appears that the isoforms do not have identical roles in papilloma development. Interestingly, apoptosis, a cellular process associated with nonsteroidal anti-inflammatory drug-induced inhibition of tumorigenesis, was not significantly altered in the epidermis or in papillomas of the COX-deficient mice. Thus, both COX-1 and COX-2 have roles in keratinocyte differentiation, and we propose that the absence of either isoform causes premature terminal differentiation of initiated keratinocytes and reduced tumor formation.