Nucleotides play a significant role in many physiologic functions, ranging from the encoding of genetic information to signal transduction. Accumulating evidence from recent animal and clinical studies supports the original notion that nucleotides are semi-essential dietary nutrients. The development of a new technique, total potentially available nucleosides, to accurately quantify the nucleotide content in various biological fluids has allowed investigators to properly assess the importance of nucleotide function and availability in various conditions. Data from animal studies indicate that exogenous nucleotides produce beneficial gastrointestinal and immunologic effects, especially during times of rapid growth, when nucleotide availability may be low. Infant studies confirm these findings and are presented in this review. Regulatory agencies are currently using this information collectively to support the rationale for the supplementation of infant formulas with higher amounts of nucleotides.