A splice variant of cyclooxygenase-1 (COX-1), COX-1b (previously termed as COX-3), has been identified in canine tissues as an acetaminophen-sensitive isoform, but the sequence of COX-1b mRNA and the encoded protein are not known in rats. We cloned and sequenced rat COX-1b mRNA from cerebral endothelial cells. Sequence analysis indicated that the 98-base pair intron-1 of COX-1 gene remains unprocessed in the COX-1b mRNA, causing a frameshift mutation and a 127-amino acid open reading frame with no sequence similarity with known cyclooxygenases. Transient and permanent transfection of COS-7 cells with a vector containing the rat COX-1b cDNA resulted in synthesis of a protein of the expected size. We generated an affinity-purified polyclonal antibody against the rat COX-1b protein. Western blot analysis of rat tissues using this antibody demonstrated the likely existence of rat COX-1b protein in vivo with the highest expression in heart, kidney, and neuronal tissues. Our results on both stable and on transiently transfected COS-7 cells suggest that rat COX-1b does not have cyclooxygenase activity and does not have any effect on the inhibition of prostaglandin production by acetaminophen. Because this protein has a completely different amino acid sequence than COX-1 and COX-2 and it does not have cyclooxygenase activity, we suggest a name cyclooxygenase variant protein to distinguish it from the known prostaglandin-synthesizing cyclooxygenase isoforms.