A myoglobin (Mb) film was successfully assembled layer-by-layer with zirconia formed by vapor-surface sol-gel deposition on solid surfaces. The method involved vaporization of zirconium butoxide (Zr(OBu)(4)) into thin layers of water, forming ZrO2 sol-gel layers after hydrolysis of Zr(OBu)(4). Mb was assembled by adsorption alternated with the ZrO2 layers. The {ZrO2/Mb}(n) layer-by-layer films at pyrolytic graphite electrodes demonstrated a pair of well-defined and nearly reversible CV peaks at about -0.34 V vs SCE, characteristic of Mb Fe(III)/Fe(II) redox couple, and used to catalyze reduction of oxygen and hydrogen peroxide. FTIR spectroscopy and comparison studies with {ZrO2/catalase}(n) films suggest that Mb in the zirconia sol-gel films essentially retains its native structure. Compared with Mb layer-by-layer films assembled with ZrO2 nanoparticles, {ZrO2/Mb}(n) films showed larger numbers of electroactive bilayers and higher maximum surface concentration of electroactive Mb, which may be related to the unique structure of ZrO2 sot-gel with higher porosity. (c) 2006 Elsevier B.V. All rights reserved.