Dental tissues contain regular microscopic structures believed to result from periodic variations in the secretion of matrix by enamel- and dentine-forming cells. Counts of these structures are an important tool for reconstructing the chronology of dental development in both modern and fossil hominids. Most studies rely on the periodicity of the regular cross-banding that occurs along the long axis of enamel prisms. These prism cross-striations are widely thought to reflect a circadian rhythm of enamel matrix secretion and are generally regarded as representing daily increments of tissue. Previously, some researchers have argued against the circadian periodicity of these structures and questioned their use in reconstructing dental development. Here we tested the periodicity of enamel cross-striations--and the accuracy to which they can be used--in the developing permanent dentition of five children, excavated from a 19th century crypt in London, whose age-at-death was independently known. The interruption of crown formation by death was used to calibrate cross-striation counts. All five individuals produced counts that were strongly consistent with those expected from the independently known ages, taking into account the position of the neonatal line and factors of preservation. These results confirm that cross-striations do indeed reflect a circadian rhythm in enamel matrix secretion. They further validate their use in reconstructing dental development and in determining the age-at-death of the remains of children whose dentitions are still forming at the time of death. Significantly they identify the most likely source of error and the common difficulties encountered in histological studies of this kind.