This report describes lipophilic conjugates of 25-kDa polyethylenimine (PEI) designed to provide better vectors for nucleic acid transfection. Conjugation of cholesterol is known both to improve transfection properties of PEIs and to reduce their cytotoxic effect. However, extensive modification would significantly decrease the polymer overall positive charge, resulting in less efficient condensation and poor delivery of nucleic acids into cells. In this study we tried to find the optimal modification extent of the PEI carrier, 25 kDa PEI, with cholesterol. Conjugates of PEI having a different number of cholesterol residues attached to their amines (5-20% of amines were modified) were synthesized. The conjugates were tested in vitro in transfection experiments, which have been carried out in the presence and in the absence of 10% FBS. Transfection efficiency studies were performed using different types of biologically active nucleic acids: single-stranded oligonucleotide, plasmid expressing vector and small interfering RNA duplex, under conditions close to those routinely applied for each type of delivered nucleic acid. PEI-cholesterol conjugates were applied at concentrations below the level at which the cytotoxic activity was observed. Results of this study demonstrate that efficient transfection and lower toxicity of the compounds is achieved at 1% modification of amino groups of the polymer.