The new substrates 4-thiouridine and 4-thiothymidine were proposed for spectrophotometric measurement of the activity of uridine (UP) and thymidine (TP) phosphorylases. At pH 7.5, 4-thiouridine has an absorbance maximum at 330 nm, and the difference in extinction coefficient (Δε) between 4-thiouridine and 4-thiouracil is 3000-1cm-1. 4-Thiouridine proved to be a good substrate for UP: the Michaelis (KM) and catalytic (kcat) constants were estimated respectively at 130 μM and 49 s-1at 25°C. Even a greater Δε (5000 M-1cm-1at 336 nm) was observed for the 4-thiothymidine/4-thiothymine pair.