The distribution of anionic groups in fibres, fines, the colloidal fraction, and the dissolved fraction of thermomechanical pulp (TMP) suspensions was determined. The influence of extraction, alkaline treatment, and peroxide bleaching of spruce TMP were also studied. Spruce TMP was extracted with hexane, treated with alkali, or bleached with peroxide. Suspensions were made at pH 5.5 or 8, and fractionated into long fibres, large fines, small fines, a colloidal fraction, and a dissolved fraction. The surface and total charge of the fractions were determined separately by polyelectrolyte titration. To determine the origin of the charges, the contents of fatty acids, resin acids, and acidic units in polysaccharides in the different fractions were determined by gas chromatography. Extraction of TMP with hexane prior to fractionation increased the measured total acid surface charge of the fibres. The removal of wood resin probably uncovered some anionic groups on the fibre surface, or improved the penetration of the polymers into the pores of the fibres. The total charge, determined with polybrene titration, of the fines and the colloidal fraction was lower when the resin had been removed, while the surface charge, determined with poly-DADMAC, was not greatly affected. Alkaline treatment of the TMP increased both the total charge and the surface charge of the fibres and fines, mainly because of demethylation of pectins. Alkaline treatment also increased the total and surface charge of the dissolved substances, due to the release of pectic acids into the water phase. Alkaline peroxide bleaching further increased the total and surface charge of fibres and dissolved substances, most likely due to oxidation of lignin. The total charge and the surface charge of the colloidal substances, consisting mainly of wood resin, were only slightly affected by alkaline treatment and peroxide bleaching. The anionic charge in TMP suspensions originated mainly from free uronic acids in the xylans, arabinogalactans, and pectic acids. The contribution from the fatty and resin acids was substantial only for the colloidal fraction.