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Enzymatic incorporation of deoxynucleoside 5'-triphosphate bearing the photocleavable protecting group is a useful method for the preparation of photocaged oligodeoxynucleotides. Here, we describe the synthesis of new photocaged deoxynucleoside triphosphates N 1-(2-nitrobenzyl)-deoxypseudouridine triphosphate (d NB ΨTP) and N 1-(6-nitropiperonyloxymethyl)-deoxypseudouridine triphosphate (d NPOM ΨTP). We successfully synthesized d NB ΨTP and d NPOM ΨTP and applied them to enzymatic synthesis of photocaged oligonucleotides. In addition, we also synthesized phosphoramidites of N 1-(2-nitrobenzyl)- and N 1-(6-nitropiperonyloxymethyl)-deoxypseudouridine to enable chemical synthesis of photocaged oligonucleotides incorporating them. The photocleavable 2-nitrobenzyl and 6-nitropiperonyloxymethyl in oligonucleotides were cleaved by irradiation at 365 nm for 30 and 10 s, respectively. We also studied the enzymatic incorporation of d NB ΨTP and d NPOM ΨTP using the Klenow fragment exo - . As a result, it was clarified that d NPOM ΨTP could be incorporated to oligonucleotide 193 times more efficiently than d NB ΨTP, as judged by V max / K m . We also performed the incorporation of at least eight d NPOM Ψ residues in a 35-mer oligodeoxynucleotide. It has also been revealed that the oligodeoxynucleotides incorporating photocaged deoxypseudouridine were useful for photocontrol of DNA triplex formation.