Peptide nucleic acid (PNA) is a DNA analogue in which the negatively charged sugar phosphate backbone has been substituted by uncharged N-(2-aminoethyl)glycine units. The study of a PNA-DNA duplex and the corresponding DNA-DNA duplex gives a unique opportunity to compare two polyelectrolytes with virtually identical geometry but greatly different linear charge density. The results provide a basis for a study of the applicability of the Poisson-Boltzmann (PB) and counterion condensation (CC) theories. UV and circular dichroism spectroscopy as well as isothermal titration calorimetry (ITC) have been used to study the effect of different ions on the stability and conformation of PNA-DNA, PNA-PNA, and DNA-DNA duplexes having the same base sequences. Cations in general destabilize both antiparallel (N/3’) and parallel (N/5’) PNA-DNA duplexes whereas they stabilize the DNA-DNA duplex. Studies on the effect of monovalent salt such as NaCl on T-m were carried out over a wide range of salt concentrations (0.01 to 5 M). The decrease in the T-m of the N/3’ PNA-DNA duplex with increasing ionic strength in the range of concentrations of 0.01 to 0.5 M, where electrostatic effects predominate, is explained in terms of counterion release upon duplex formation in contrast to the counterion association accompanying the formation of a DNA duplex. The uncharged PNA-PNA duplex shows no significant destabilization in this concentration range. The higher stability of the N/3’ PNA-DNA compared to the DNA-DNA duplex (Delta Delta G similar to-7 kcal/mol) is ascribed to more favorable entropic contributions consistent with the counterion release that accompanies the PNA-DNA duplex formation. At high salt concentration (>1 M), where electrostatic contributions saturate, similar trends in the decrease in T-m, were observed for the three types of duplexes irrespective of their backbone charges. The destabilizing effects of a series of Na salts with various monovalent anions on N/3’ PNA-DNA and PNA-PNA duplexes were found to follow the Hofmeister series, emphasizing the importance of the hydrophobic interaction between nucleobases for the stability of the PNA complexes in high salt concentration.