Polysialic acid is a developmentally regulated carbohydrate attached to the neural cell adhesion molecule, N-CAM, and abundant in embryonic tissues. There is increasing evidence that polysialic acid reduces N-CAM adhesion, thereby promoting neurite outgrowth and cellular mobility. It has been shown that two enzymes, polysialyltransferase, PST, and sialyltransferase X, STX, form polysialic acid on N-CAM. However, it is not known how these two enzymes contribute to polysialylation. In order to determine how the expression of PST and STX leads to polysialic acid synthesis during mouse development, the expression of PST and STX transcripts were evaluated by Northern blot analysis, competitive reverse transcription-polymerase chain reaction and in situ hybridization, and those results were correlated to the expression of polysialic acid. The results obtained by these analyses demonstrated that both PST and STX transcripts were barely detected at embryonic day 8 (E8) but increased after E9. PST and STX transcripts were present in substantial quantity between E11 and E15, coinciding with the period when maximum synthesis of polysialic acid is required. Ten days after birth, the level of STX transcript declined substantially, whereas the level of PST transcript only gradually declined and persisted in the adult brain. These results, taken together, strongly suggest that PST and STX coordinately synthesize polysialic acid during development. At the same time, they are expressed differentially in tissue-specific and cell-type-specific manners, suggesting that PST and STX may have distinct roles in development and organogenesis.