Objective: To examine how eicosanoid levels in menstrual effluent of adolescents within three years of menarche relate to the severity of menstrual pain. Design: Prospective cohort study. Setting: Community teaching hospital. Population: Adolescents within three years after menarche. Methods: Participants provided a menstrual effluent sample between 4 and 30 months after menarche. Eicosanoids and oxylipins concentrations were measured in the menstrual effluent. We compared effluent concentrations of participants with dysmenorrhea (>0 on a 0-10 scale, n=33) to age-matched pain-free controls (0 on a 0-10 scale, n=18). Main Outcome Measures: Eicosanoid and oxylipins concentrations in menstrual effluent. Results: Participants with dysmenorrhea had higher PGF2α (4.5 [1.6, 8.9] ng/ml, p=0.014) than controls (1.1 [0.07, 4.4] ng/ml). However, differences in PGE2 (7.1 [2.6, 10.1] vs. 3.5 [1.0, 5.1], p=0.053) and 12-HETE (36.3 [23.7, 60.7] vs. 29.6 [13.4, 51.5], p=0.305) were not significant. The correlations between PGF2α (r=0.37, p=0.004) or PGE2 concentration (r=0.28, p=0.046) and menstrual pain intensity were moderate to small. Overall, there were positive correlations between menstrual volume and eicosanoid concentrations (r’s >0.4, p’s< 0.001). Intriguingly, dysmenorrhea participants taking analgesics had a high total content of PGF2α (66.2 [43.0,164.7]) compared to controls (19.1 [6.0,47.5], p=0.04). LC-MS/MS revealed higher concentrations of 12-HETE, 14,15-EET, 15-HETE, 18cdLTB4, LTB4, and PGF2α—and lower 6-kPGF1α—in the effluent of dysmenorrhea participants compared to controls. Conclusions: Elevated PGF2α in adolescents with dysmenorrhea, modest correlations between prostaglandin concentrations and menstrual pain, and the identification of additional oxylipins suggest that inflammatory processes beyond the prostaglandin pathway may contribute to dysmenorrhea.