The use of reversed-phase HPLC with UV detection at 254 nm for the separation of stereoisomers of nucleoamino acids, namely, pyrimidyl and purinyl derivatives of alanine, after premodification with o-phthalic aldehyde and N-acetyl-L-cysteine was studied. The use of 0.01 M phosphate buffer (pH 7.0) containing 5% acetonitrile as a mobile phase resulted in satisfactory separation. The range of the detectable amounts of nucleoamino acids was 0.08-1.0 nmol. This method was used for monitoring the formation of stereoisomers in the hydrolysis of N-phenylacetylcytosinylalanine.