An empirical formula for thermal stability (Tm) prediction of PNA/DNA duplexes has been derived. The model is based on the Tm as calculated for the corresponding DNA/DNA duplex employing a nearest neighbour approach, by including terms for the pyrimidine content and length of the PNA to take into account the increased thermostability of PNA/DNA hybrids and the asymmetry of the PNA-DNA heteroduplex. The predictive power of the Tm prediction formula was challenged with an independent data set not used for model building. The Tm of >90% of the sequences was predicted within 5 K; 98% of the predicted Tms differ by not more than 10 K from the experimentally determined Tm.