We tested several sample pre-treatment protocols for the study of oxygen isotope ratios in the phosphate phase of mammalian enamel of ten different fossil samples. We investigated the effect of different pre-treatment methods and the duration of the hydrogen fluoride treatment on enamel samples from skeletal phosphate with known δ18O values. The samples had been measured previously, so we could compare the ratios measured in our laboratory with the previous values to choose the best chemical preparation procedure. Four pre-soaking methods and two different time intervals of 2 mol dm−3 hydrogen fluoride treatment were compared during our experiments. In our experimental conditions, the distilled water wash and the 6 h of soaking in hydrogen fluoride gave the closest results to the expected δ-values. The steps of the tested preparation processes were repeated at least three times on each sample, so the reproducibility of the process could be also investigated.