Applying various restriction enzymes on a specially designed 1.5 kb DNA fragment revealed that the inhibitory effects of psoralens UVA irradiation (PUVA) treatment on restriction endonuclease activities are caused by recognition inhibition. In this study restriction enzymes that have a 5′-TpA sequence at the cleaving site (KpnI, XbaI, PmeI and DraI), and the noncleaving site (PacI) in recognition sites, or have two 5′-TpA sequences at the recognition site, and a nonspecific sequence between the recognition and the cleaving sites (BciVI), were inhibited by PUVA treatment. Most of the other restriction enzymes used in this study, which do not have a 5′-TpA sequence at their restriction site, were not inhibited by PUVA treatment, although a 5′-TpA sequence is located adjacent (SmaI) or very close (BamHI, SacI and PstI) to the recognition and cleaving sites for these enzymes. Because SphI, which does not have 5′-TpA at its restriction site, was strongly inhibited by PUVA treatment, the 5′-CpA sequence is suggested to be a new binding site of psoralens after UVA irradiation.