Background/Question/Methods C4 photosynthesis is a major ecological and evolutionary success in terrestrial ecosystems, particularly within the grass family. C4 grasses account for ~25% of global primary productivity, despite the fact that only ~3% of land plants use C4 photosynthesis, which makes it vital to understand when and why they evolved. Unfortunately, grass pollen is morphologically indistinct, making pollen analysis a blunt instrument for studying C4-grass evolution using geological samples. Studies have investigated the timing of C4 evolution using molecular tools and d13C records from n-alkanes, ungulate teeth, and paleosols, but they yield disparate results. Molecular clocks suggest that C4 grasses first evolved during the Oligocene, coincident with a decline in pCO2 from >1000 to <500 ppm. In contrast, d13C-based approaches do not reliably detect the presence of C4 grasses until the middle Miocene, indicating they were previously uncommon on the landscape. To detect the initial evolution of C4 photosynthesis in the grass family, we devised a technique that reliably distinguishes C4 from C3 grass pollen: Single Pollen Isotope Ratio AnaLysis (SPIRAL). We used SPIRAL to determine the d13C composition of single grains of grass pollen (100/sample) in geological samples from Asia and Europe spanning the late Oligocene to middle Miocene. Results/Conclusions We used an optimal threshold value (-19.2‰, Nelson et al., 2007) adjusted for small (< 1‰) temporal variations in atmospheric d13C to distinguish C3/C4 ratios. Initial results suggest that C4 grasses produced 20-40% of grass pollen during the late Oligocene through middle Miocene at our sites, which supports molecular data that suggests C4 grasses had evolved by at least the late Oligocene. The results are thus consistent with the supposition that the declining pCO2 during the Oligocene was the likely primary driver of C4 evolution. However, estimates for the timing of C4 evolution remain imprecise. The earliest origin of C4 might have occurred 25-32 Ma on the basis of molecular data, and we do not have SPIRAL data from samples older than ~25 Ma. We are currently analyzing well-dated samples from additional sites spanning the late Eocene through Miocene. In addition, we are refining the C3/C4 threshold value using grass pollen from herbarium specimens. New results should help pinpoint the timing of C4 evolution. Comparison of the timing of C4 evolution with independent pCO2, paleoclimate and paleoecological records will be used to assess the factor(s) that drove the evolution and eventual dominance of C4 grasses by the late Miocene.