To further identify and characterize the nitrogen fraction of human milk, nucleotide and total nitrogen contents were determined using high pressure liquid chromatography and Kjeldahl analyses. Five lactating women were followed longitudinally. Each provided 16 milk samples (8-10 ml each) collected before and after a single nursing, and in the morning and afternoon of a single day. This collection scheme was followed at 2, 4, 8, and 12 wk postpartum. The variance pattern of nucleotides was observed to be distinct from that of total nitrogen. As the lactation period progressed from wk 2 to 12 postpartum, levels of cytidine 5' monophosphate and adenosine 5' monophosphate declined from 594 to 321 micrograms/100 ml and from 244 to 143 micrograms/100 ml, respectively, whereas levels of inosine 5' monophosphate increased from 158 to 290 micrograms/100 ml and levels of total nucleotide nitrogen remained constant. Nucleotide nitrogen accounted for approximately 0.1-0.15% of the total nitrogen content of human milk samples analyzed. Total concentration of human milk was observed to decrease as lactation progressed and to be higher in afternoon than in morning samples. The nucleotide profile of human milk was characteristically different from that of other milks commonly used an infant feeding. It is estimated that an infant consuming human milk as a principal nutrition source would ingest 1.4-2.1 mg of nucleotide nitrogen per day.