Radiogenic dental damage is thought to be the result of reduced salivary flow as well as possible direct radiogenic damage. The exact nature of the latter is still to be elucidated. We set out to assess whether there was measurable direct and immediate radiogenic damage to the collagen component of dental hard and soft tissues. A total dose of 31.5 Gy was applied to 40 human third molar teeth in vitro (cobalt 60, 6.3 Gy/day for 5 days) (group 1), 40 further third molar non-irradiated human teeth served as controls (group 2). Collagen fragments (split collagen) of mineralized tissue (a) and pulpal tissue (b) of groups 1 and 2 were isolated by ultrafiltration and pooled separately for each experimental group. Measurement of the mature collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) by high performance liquid chromatography (HPLC) was used to determine the ratio of the amount of collagen fragments from irradiated as opposed to non-irradiated teeth and assessing mineralized from pulpal tissue separately. No significant difference was found between the concentration of collagen cross-links in probes of mineralized tissue between groups 1 and 2. The concentration of HP and LP in probes of irradiated dental pulp however was significantly increased (ratio: 3.4 and 3.4 times) as compared to pooled probes from non-irradiated pulp. Irradiation does not measurably affect the collagen component in mineralized dental tissue, which may be due to the relatively low concentration of this protein in dentin and enamel. In contrast, direct and instant radiogenic damage of (extracellular matrix) pulpal tissue collagen could be demonstrated.