Prostaglandin endoperoxide H (PGH) synthases 1 and 2 are both membrane-associated proteins localized to the endoplasmic reticulum (ER) and nuclear envelope. The carboxyl terminal tetrapeptides of PGH synthases 1 and 2 are of the form -P/STEL. These sequences are similar to the -KDEL retention signal sequence characteristic of many proteins localized to the ER. To determine if the -PTEL sequence (residues 597-600) functions as an ER retention signal for ovine PGH synthase-1, we prepared and analyzed five mutants (L600N, L600R, L600V, E599Q, and delta 597), all having modifications that would be expected to alter the subcellular location of PGH synthase-1 if the -PTEL sequence were involved in ER targeting. Native ovine PGH synthase-1 and each of the five mutants were subcloned into the pSVT7 expression vector and were expressed transiently in cos-1 cells. The L600N, L600R, E599Q, and delta 597 mutants retained both cyclooxygenase and peroxidase activities. Moreover, when subjected to immunocytofluorescent staining, cos-1 cells expressing native and mutant enzymes showed similar patterns of fluorescence corresponding to ER and nuclear envelope localization. Finally, culture media bathing cos-1 cells transfected with native or mutant PGH synthases were tested for secreted PGH synthase-1 protein by Western blotting, but no PGH synthase-1 was detected in any of the culture media. Our results demonstrate that mutations in the C-terminal sequence-PTEL do not change the subcellular location of ovine PGH synthase-1. Thus, targeting of PGH synthase-1 to the ER can occur independent of its -PTEL sequence.