The objective of this study was to evaluate the effects of irradiation and surface condition on in situ remineralization of demineralized human and bovine enamel. Specimens (n = 96) obtained from 24 human molars and 24 bovine incisors were prepared. The surfaces of half of the specimens were abraded while the others remained natural. Each of the 12 human and bovine abraded and sound specimens was irradiated fractionally up to 46.5 Gy (3.1 Gy/day, 5 days/week), while the remaining samples were not irradiated. Prior to and following the demineralization (pH 5.0; 14 days) all specimens were partly covered with nail varnish (control). After in vitro demineralization 2 irradiated and 2 nonirradiated specimens were inserted into both buccal aspects of 12 intraoral appliances, which were worn by 12 persons for a period of 6 weeks. All samples were brushed twice a day with a fluoride-containing toothpaste. Mineral analyses revealed a significant influence on mineral loss (ΔZdemin), mineral gain (ΔΔZ), lesion depth (LDdemin), and lesion depth reduction (ΔLD) of the variables ‘material’ and ‘irradiation’ (p < 0.05; ANOVA). Reciprocal interactions could be observed between these two variables for ΔZdemin, ΔΔZ, and LDdemin, due to significantly reduced values found for the irradiated, abraded bovine specimens compared to the nonirradiated ones (p < 0.05; Bonferroni post hoc test). It can be concluded that neither irradiation nor abrasion influenced in vitro demineralization or in situ remineralization of the human specimens, whereas abrasion hampered demineralization in irradiated, bovine specimens.