Fetal tau and tau in paired helical filaments show similar immunoreactivities with several phosphorylation-dependent paired helical filament-polyclonals and monoclonals, suggesting that the two molecules share several distinct phosphorylated epitopes. To make clear the similarities and differences between the two, we have undertaken work to identify the in vivo phosphorylation sites in fetal rat tau. We have approached this problem by identifying phosphopeptides by means of mass spectrometry and sequencing of those phosphopeptides after modification with ethanethiol. Although remarkable heterogeneity was present, fetal tau was found to bear at most 10 phosphates at Ser-189, Ser-190, Ser-193, Ser-226, Ser-387, Ser-395, Thr-172, Thr-222, and, presumably, Ser-391 and Thr-208 (numbering is according to the longest form of rat tau; Kosik, K. S., Orecchio, L. D., Bakalis, S., and Neve, R. L. (1989) Neuron 2, 1389-1397). In contrast, adult rat tau was much less phosphorylated; only Thr-172, Ser-190, Ser-193, Thr-222, and Ser-395 were phosphorylated to a slight-to-moderate extent. All these sites except for Ser-189 and Ser-391 were followed by Pro residues. Thus, tau is an in vivo substrate for proline-directed protein kinase(s), and its phosphorylation state is developmentally regulated.