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Determining absolute ages of carbonate diagenesis, faulting, fossil formation, speleothem growth, carbonate-hosted hydrocarbon deposits, vein mineralisation and hydrothermal alteration has become increasingly accessible through LA-ICP-MS U-Pb dating of calcite, complementing traditional isotope dilution methods still applicable to certain materials (e.g., speleothems via micro-drilling). However, well-calibrated reference materials for LA-ICP-MS calcite geochronology remain scarce. Here, we characterise the Rio Maior calcite, designated ‘RioM-1’, as a potential reference material for U-Pb dating by LA-ICP-MS. Fragments (0.1 to 1 cm3) from a single scalenohedral crystal were analysed by ID-TIMS (U-Pb), LA-ICP-MS (U-Pb and 87Sr/86Sr), and SIMS (O isotopes). RioM-1 displays high U mass fraction and low, though variable, proportions of common Pb. Combined ID-TIMS analyses from two independent laboratories yielded a Tera-Wasserburg lower intercept date of 63.93 ± 0.11 Ma (2s, MSWD = 1.3, n = 16). LA-ICP-MS U-Pb data from three independent laboratories are concordant with the TIMS age, producing a pooled date of 64.10 ± 0.12/1.2 Ma (2s, MSWD = 7.7, n = 708) and an initial 207Pb/206Pb ratio of 0.85 ± 0.01. Other calcite reference materials, when normalised to RioM-1, yielded dates within uncertainty of their published values. SIMS measurements returned a mean 18O/16O of 0.002044450 ± 181 (1s) and δ18OSMOW of 19.57 ± 0.92‰ (1s), while LA-ICP-MS strontium isotope measurement yielded a mean 87Sr/86Sr of 0.708177 ± 9 (2s).