A pyruvate decarboxylase gene of Pisum sativum (PDC1) was expressed in the yeast Saccharomyces cerevisiae. The recombinant homomeric pyruvate decarboxylase (recombinant PsPDC) of subunit molecular mass 64 kDa, was soluble and had a specific enzymatic activity of 57 U/mg, which is comparable to that of native PsPDC. As revealed by electron microscopy, the native and the recombinant holoenzymes form filamentous structures. Removal of the cofactors ThDP and Mg2+ caused reversible dissociation to the lower apoenzyme oligomers. Image analysis, made from transmission electron microscopy images of negatively stained samples, showed both holoenzyme filaments to be a twisted string of building blocks with axial periods of 19.29 nm and 19.05 nm for the recombinant and native PsPDC, respectively. These values correspond to three repeating units per turn, which mass measurement by scanning transmission electron microscopy identified as tetramers. Near UV CD spectra and fluorescence spectra of the apo- and holoenzyme species differed in the same way for the native and the recombinant PsPDC proteins indicating very similar tertiary structures of both enzyme forms. The S0.5 value of pyruvate was 2 mM at 30°C for the recombinant enzyme which is twice as high as for the native protein. Like the native enzyme, recombinant PsPDC is activated by its substrate pyruvate. However, the sigmoidal behaviour in the v-S characteristics is much more pronounced, indicating that at least three substrate molecules must be bound for the decarboxylation of one molecule at maximum velocity. As revealed by stopped-flow measurements, this behaviour is reflected by the kinetic parameters of the substrate activation process.