Maternal hormones deposited in the egg can provide a powerful model for the study of maternal effects. The differential amount of maternal hormones in the yolk of freshly laid eggs is assumed to represent differential maternal allocation. However, some evidence suggests that these amounts do not reflect maternal allocation that in fact takes place before ovulation. We compared the amounts of a wide array of gonadal steroids and their metabolites in the yolk of pre-ovulatory follicles with those of freshly laid eggs of rock pigeons using mass spectrometry. We found that between the follicle and egg stages the levels of progesterone increase whereas androstenedione and testosterone decrease in which the strength of decrease was dependent on the laying order of the egg. For conjugated estrone the change between follicle and egg differed in direction for first and second laying position yielding a significant interaction effect. For conjugated testosterone the interaction did not reach but was close to significance. This extremely early steroid metabolism was not due to maternal enzymes in the yolk as indicated by incubation of pre-ovulatory yolks treated with proteinase-K, a protein digesting enzyme. The results have significant consequences for the functional and evolutionary interpretation as well as experimental manipulation of hormone-mediated maternal effects.