In a prospective cohort study on atopy in childhood, three skin prick tests (SPT) were performed in 1135 primary schoolchildren over a 2–year period. We studied the development of average sizes of allergen wheals in reactive children and of average sizes of histamine wheals in all children, and we investigated the influence of choice of SPT criterion on the prevalence and incidence of sensitization. A year-to-year increase of average allergen wheal sizes as well as of average histamine wheal sizes was observed. These increases seemed to be related to the natural growth of the children. Furthermore, an observer bias effect of the person who did the pricking on average wheal sizes was found. There were considerable differences in estimations of prevalences and incidences depending on the definition of SPT criterion. Estimations using a criterion based solely on the allergen wheal size were affected by variation of allergen wheal sizes caused by growth and observer effects. The ratio criterion was not influenced by observer effects, nor did it seem to be affected by aging effects. In conclusion, our data suggest that the ratio criterion, which relates the size of the allergen wheal to the size of the histamine wheal, is the most appropriate SPT criterion because it is not affected by growth of allergen wheals in childhood and it appears to compensate for possible observer bias.