Coral bleaching is of increasing concern to reef management and stakeholders. Thus far, quantification of coral bleaching tends to be heavily reliant on the enumeration of zooxanthellae, with less emphasis on assessment of photosynthetic or physiological condition, these being often assessed separately by techniques such as liquid chromatography. Traditional methods of enumeration using microscopy are time consuming, subjected to low precision and great observer error. In this study, we presented a method for the distinction of physoiological condition and rapid enumeration of zooxanthellae using flow cytometry (FCM). Microscopy verified that healthy looking/live versus damaged/dead zooxanthellae could be reliably and objectively distinguished and counted by FCM on the basis of red and green fluorescence and light scatter. Excellent correlations were also determined between FCM and microscopy estimates of cell concentrations of fresh zooxanthellae isolates from Pocillopora damicornis. The relative intensities of chlorophyll and b-carotene fluorescences were shown to be important in understanding the results of increased cell counts in freshly isolated zooxanthellae experimentally exposed to high temperatures (34, 36, and 388C) over 24 h, with ambient temperature (298C) used as controls. The ability to simultaneously identify and enumerate subpopulations of different physiological states in the same sample provides an enormous advantage in not just determining bleaching responses, but elucidating adaptive response and mechanisms for tolerance. Therefore, this approach might provide a rapid, convenient, and reproducible methodology for climate change studies and reef management programs. ' 2012 International Society for Advancement of Cytometry Key terms bleaching response; zooxanthellae; chlorophyll; flow cytometry; microscopy GLOBAL-scale coral bleaching has been occurring with increasing frequency over the past two decades (1,2). High seawater temperatures and solar irradiance are recognized as the major threats to coral ecosystems, and high incidences of these are expected to occur with increased frequency with climate change (2-4). Coral bleaching is caused by the degradation of photosynthetic pigments and/or ''in situ'' degradation of symbiotic dinoflagellates of the genus Symbiodinium (zooxanthellae), coupled with the expulsion of zooxanthellae in more severe instances (5-7). A more comprehensive understanding of the changes in symbiont densities and photosynthetic pigment concentrations in corals caused by stress will provide a useful means of distinguishing both in assessing the health and in bleaching status of corals.Zooxanthellae contain photosynthetic pigments chlorophyll a (chl a) and chlorophyll c2, which serve to capture light, whereas accessory pigments such as carotenoids provide photoprotective-type functions (8-12). Zooxanthellae densities and their pigment concentrations have been reported as reliable and replicable methods for the evaluation of the status of coral bleaching (13). Prev...