Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, while conversion of AMP to adenosine is largely mediated by soluble enzymes. The identity of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine while soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'NT and AP activity and lower adenosine metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant as supplementation with AMP inhibited while selective pharmacologic inhibition of 5'NT enhanced Toll-like receptor-mediated TNFα production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine metabolizing enzymes may benefit this vulnerable population.