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The Amazon rain forest is a major global isoprene source, but little is known about its seasonal ambient concentration patterns. To investigate the environmental and phenological controls over isoprene seasonality, we measured isoprene mixing ratios, concurrent meteorological data, and leaf area indices from April 2014 to January 2015 above a rain forest in the central Amazon, Brazil. Daytime median isoprene mixing ratios varied throughout the year by a factor of two. The isoprene seasonal pattern was not solely driven by sunlight and temperature. Leaf age and quantity also contributed to the seasonal variations of isoprene concentrations, suggesting leaf phenology was a crucial variable needed to correctly estimate isoprene emissions. A zero-dimensional model incorporating the estimated emissions, atmospheric boundary layer dynamics, and air chemistry was used to assess the contributions of each process on the variability of isoprene. Surface deposition was an important sink mechanism and accounted for 78% of the nighttime loss of isoprene. Also, chemical reactions destroyed isoprene and during 6:00 to 18:00 h local time 56, 77, 69, and 69% of the emitted isoprene was chemically consumed in June, September, December, and January, respectively. Entrainment fluxes from the residual layer contributed 34% to the early-morning above-canopy isoprene mixing ratios. Sensitivity analysis showed that hydroxyl radical (HO) recycling and segregation of isoprene HO played relatively lesser roles (up to 16%) in regulating ambient isoprene levels. Nitric oxide (NO) levels dominated isoprene chemical reaction pathways associated with consumption and production of HO under low-NO and high volatile organic compound (VOC) conditions. While surface deposition and oxidative processes altered isoprene levels, the relative importance of these factors varied seasonally with leaf phenology playing a more important role.