Complexes of DNA with cationic lipids and cationic polymers are frequently used for gene transfer. Extracellular interactions of the complexes with anionic glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of GAGs with carrier DNA complexes have been studied using tests for DNA relaxation (ethidium bromide intercalation), DNA release (electrophoresis), and transfection (pCMVbGal transfer into RAA smooth muscle cells). Several cationic lipid formulations (DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured dendrimer, polyethylene imines 25 and 800 kDa, polylysines 20 and 200 kDa) were tested. Polycations condensed DNA more effectively than monovalent lipids. Hyaluronic acid did not release or relax DNA in any complex, but it inhibited transfection by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by other carriers was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin sulfates B and C) completely blocked transfection, except in the case of liposomes with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these events were not prerequisites for the inhibition of transfection. Furthermore, preliminary results suggest that cell surface GAGs, particularly heparan sulfate, inhibit gene transfer by cationic lipids and polymers.