Poly(ADP-ribose) is a variably sized homopolymer synthesized from NAD as a covalent adduct to chromosomal proteins; its synthesis is catalyzed by the enzyme poly(ADP-ribose)synthetase (pADPRS). Using an assay to estimate the pADPRS levels during various phases of both in vivo and in vitro limb mesenchymal cell development, we report that the level of pADPRS undergoes a substantial change as limb cells differentiate into muscle or cartilage. This change involves a threefold transient increase in the level of pADPRS per unit DNA which is coincident with the initiation of specific phenotypic expression. These fluctuations are observed for both chondrogenic and for myogenic events. Such a transient increase in pADPRS levels seems to be characteristic of differentiating cells but is not observed in cells which have already differentiated. These observations establish a correlation between pADPRS levels and chick limb mesenchymal cell differentiation both in vivo and in vitro and suggest that previous quantification of in situ ADP-ribosylation activity reflects alterations in pADPRS levels. Based on the information reported here and by others, a speculative hypothesis is put forth to explain the role of poly(ADP-ribose)synthetase in developmental events.