BACKGROUND AND AIM: Higher exposure to traffic-related air pollution (TRAP) is related to lower fertility, with specific adverse effects on the ovary. Epigenetics, specifically DNA methylation (DNAm), may underlie this association but given its tissue specificity, exploring potential mechanisms in toxicological-relevant target tissues is essential. Our objective was to explore the relation between TRAP exposure and follicular fluid (FF) DNAm. METHODS: Our study included 62 women undergoing in vitro fertilization at a fertility center in Boston, Massachusetts (2005-2015). TRAP exposure was defined using a spatiotemporal model to estimate residence-based daily nitrogen dioxide (NO2) concentrations in the 3 months prior. FF was collected during oocyte retrieval and DNAm levels were profiled using the Infinium MethylationEPIC BeadChip. We estimated epigenetic age via an existing granulosa cell clock and other common epigenetic clocks. We used linear regression to test whether accelerated epigenetic age, or differential DNAm across the genome, was associated with NO2 exposure while adjusting for surrogate variables and other confounders. RESULTS: Epigenetic age as measured using the granulosa cell clock was moderately correlated with reported age (r=0.48,p<0.001). Other epigenetic age estimators performed poorly in the FF. There was no association between NO2 exposure and accelerated epigenetic aging (β=-0.05 95%CI -1.20,1.10). NO2 exposure was associated with 8 differentially methylated regions and 22 differentially methylated positions, after adjusting the significance threshold for 704,169 tests. The genes annotated were enriched for GATA1 transcription factor binding and for DNA-methyltransferase activity (q-val<0.05). Some of the differentially methylated genes, including KCNQ1, VRK1, GPER, MLH1, and PLET1, have been linked to reproductive and fertility outcomes. CONCLUSIONS: Our study suggests that differences in FF DNAm could be a plausible mechanism potentially linking TRAP exposure to ovarian dysfunction. Future work to improve assessment of epigenetic age in reproductive tissues, such as FF, is warranted. KEYWORDS: Air pollution, Epigenetics, Ovary, Fertility