Solvent effect on the acid-base behavior of polycytidylic acid [poly(C)] is studied by means of potentiometric and spectrometric (UV and CD) procedures. Low-polarity biological environments are mimicked by using a 30% (v/v) dioxane-water mixture. Experiments performed in this medium are compared with previously reported results in aqueous solution in order to determine changes in both thermodynamic and structural aspects associated with modifications of the biomolecular surroundings. Potentiometric and spectrometric studies reveal the presence of a nonlinear polyelectrolytic effect associated with the protonation of poly(C) in the hydroorganic mixture, different from the analogous effect in aqueous solution. The curve resolution method alternating least squares is applied to the poly(C) spectrometric data. Concentration profiles and spectra of both deprotonated poly(C) and half-protonated [poly(C).poly(CH+)] are thus obtained. The fully protonated species poly(CH+) precipitates in the hydroorganic medium at pH values lower than 4. Evidence for the ordered structure of both poly(C) and [poly(C).poly(CH+)] species is seen through the comparison of the macromolecule spectra with those of the cytidine-3',5'-cyclic monophosphate monomer. Polarity decreases around the macromolecule produce significant hypochromicity in the CD spectra and hyperchromicity in the UV spectra, both signs of a disordering effect in the macromolecular structure due to the weakening of base stacking interactions.