Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in virions was primarily and constitutively phosphorylated on serine(s) in a single tryptic phosphopeptide TP1. By two-dimensional thin-layer electrophoresis and chromatography analysis of tryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is serine249. Interestingly, when serine249 was deleted or mutagenized alternate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (delta 208-236). Mutagenesis of proline250 to alanine abrogated phosphorylation at serine249 suggesting that proline250 is essential for the primary phosphorylation of the P protein. Conceivably, serine249 phosphorylation is mediated by a proline-directed protein kinase. This finding is unusual because a majority of the P proteins from other negative-strand RNA viruses have been shown to be phosphorylated primarily by casein kinase II. Our results demonstrate that the P protein has a strong potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all potential sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural integrity of the Sendai virus P protein.