The sequential extraction procedure developed by Tessier et al. was used in sludge anaerobic digestion to determine the heavy metal speciation. Sludge samples were taken every three days to investigate humic substances, VS/TS, pH, VFA, alkalinity, ammonia, the total content of Zn, Pb, Cu, Ni, and Cr, and also their distribution into EXCH, CARB, FeMnOx, OMB and RESI fractions. Results showed that, (1) Heavy metals were concentrated during the anaerobic digestion process. The concentration of all five kinds of heavy metals increased by about 50%. (2) The distribution of these heavy metals differed. (3) High-solid anaerobic digestion much or less increased the bioavailability of Cu, Zn, Ni and Cr while decreased the bioavailability of Pb. (4) There were significant degrees of correlation between heavy metal fractions and changes of some selected parameters (for example, pH, VS/TS, and VFA content). Except for Pb, the contents of total mobile fractions for Cu, Zn, Ni, Cr could be predictable from its total content. (C) 2012 Elsevier Ltd. All rights reserved.