Abstract Trimethylammonium-2-hydroxypropyl-(TMAHP) spacer was introduced into heparin (H) and the prepared films were characterized by elemental analysis, NMR, SEC-MALS, TG/DTG/DTA, AFM and mechanical tester. When quaternized at the ratio of H/NaOH/alkylating agent/H2O=0.1–1/0–2/0.1–3/50–500mmol, H was substituted at A6 and A3 positions. The formation of double-substituted structures by substitution of free hydroxyl group of the previously introduced TMAHP substituent is evident. In the absence of NaOH (H/GTMAC/H2O=1:1:500) the most drastic decrease of Mn to 8.639kg/mol and Mw/Mn at 1.48 was observed in comparison to H (Mn=9532g/mol with Mw/Mn=1.38). The film mechanical properties were better on H (E=4030MPa; σb=65MPa; ɛb=4.6%) than on quaternized specimens (E=2500–3340MPa; σb=25–40MPa; ɛb=1.7–1.8%). The AFM images did not prove relation between mechanical properties and surface shape.