Abstract: Phosphonylmethoxyethyl)adenine (1),PMEA,an acyclic nucleotide withbroad-spectrum antiviral activity was synthesized with some modifications of Holy's procedure.Simutaneously,an N-3 regioisomer(2)of PMEA and a by-preduct, formaldehyde di-2-(9-adenyl)ethyl acetal(7)were seperated by silica gel chromatography in the ratio of 50:10:1.Compound(2)and(7) are new compounds that we have not yet found in literatures. The structure of them weredetermined with ¹HNMR,2DNMR, MS and Spot test.Antiviral test showed that N-3 isomer(2)completely lost activity against both HIV-1 and HSV-1 in vitro. It seems that regiospecificity of theacyclic nucleotide structure is important for antiviral activity.