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Isotopic analyses of cactus spines grown serially from the apex of long-lived columnar cactuses may be useful for climatological and ecological studies if time series can be reliably determined from spines. To characterize the timescales over which spines may record this information, we measured spine growth in saguaro cactus over days, months, and years with time-lapse photography, periodic marking, and postbomb radiocarbon dating and then analyzed isotopic variability over these same timescales and compared these measurements to local climate. We used daily increments of growth, visible as transverse bands of light and dark tissue in spines, as chronometers to develop diurnally resolved delta C-13 and delta O-18 records from three spines grown in series over a 70 day period. We also constructed a 22 year record of delta C-13 variations from spine tips arranged in chronological sequence along the side of a 4 m tall, single-stemmed saguaro. We evaluated two mechanisms potentially responsible for daily, weekly, and annual variability in delta C-13 values of spines; both related to vapor pressure deficit (VPD). Our data suggest that stomatal conductance is unlikely to be the determinant of delta C-13 variation in spines. We suggest that either VPD-induced changes in the balance of nighttime- and daytime-assimilated CO2 or mesophyll-limited diffusion of CO2 at night are the most likely determinant of delta C-13 variation in spines. Intra-annual and interannual variability of delta O-18 in spine tissue appears to be controlled by the mass balance of O-18-depleted water taken up after rain events and evaporative enrichment of O-18 in tissue water between rains. We were able to estimate the annual growth and areole generation rate of a saguaro cactus from its 22 yearlong isotopic record because VPD, rainfall, and evaporation exhibit strong annual cycles in the Sonoran Desert and these variations are recorded in the oxygen and carbon isotope ratios of spines.