L-Serine dehydratase from Lactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM L-cysteine in 50 mM phosphate buffer. M(r) = 150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M(r) = 40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K(m) for L-serine was 65 mM. Fe++ was required for the enzymatic activity, and the apparent K(m) value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45-degrees-C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal x mol-1 for temperature values more than and less than 35-degrees-C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5'-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027-mu-moles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.