Inhibitors of cyclooxygenase (COX)-2 prevent suppression of aquaporin-2 and reduce polyuria in the acute phase after release of bilateral ureteral obstruction (BUO). We hypothesized that BUO leads to COX-2-mediated local accumulation of prostanoids in inner medulla (IM) tissue. To test this, rats were subjected to BUO and treated with selective COX-1 or COX-2 inhibitors. Tissue was examined at 2, 6, 12, and 24 h after BUO. COX-2 protein abundance increased in IM 12 and 24 h after onset of BUO but did not change in cortex. COX-1 did not change at any time points in any region. A full profile of all five primary prostanoids was obtained by mass spectrometric determination of PGE₂, PGF_2α, 6-keto-PGF_1α, PGD₂, and thromboxane (Tx) B₂ concentrations in kidney cortex/outer medulla and IM fractions. IM concentration of PGE₂, 6-keto-PGF_1α, and PGF_2α was increased at 6 h BUO, and PGE₂ and PGF_2α increased further at 12 h BUO. TxB₂ increased after 12 h BUO. 6-keto-PGF_1α remained significantly increased after 24 h BUO. The COX-2 inhibitor parecoxib lowered IM PGE_2, TxB₂, 6-keto-PGF_1α, and PGF_2α below vehicle-treated BUO and sham rats at 6, 12 and, 24 h BUO. The COX-1 inhibitor SC-560 lowered PGE₂, PGF_2α, and PGD₂ in IM compared with untreated 12 h BUO, but levels remained significantly above sham. In cortex tissue, PGE₂ and 6-keto-PGF_1α concentrations were elevated at 6 h only. In conclusion, COX-2 activity contributes to the transient increase in prostacyclin metabolite 6-keto-PGF_1α and TxB₂ concentration in the kidney IM, and COX-2 is the predominant isoform that is responsible for accumulation of PGE₂ and PGF_2α with minor, but significant, contributions from COX-1. PGD₂ synthesis is mediated exclusively by COX-1. In BUO, therapeutic interventions aimed at the COX-prostanoid pathway should target primarily COX-2.