The enzymatic method for cholesterol determination can use either an endpoint or a kinetic method. Not much is known concerning the properties (K(m) and V(max)) of the commercial enzyme for the kinetic method. We measured the K(m) and V(max) of Brevibacterium, Streptomyces, Pseudomonas fluorescens, and Cellulomonas cholesterol oxidase. Brevibacterium gave the highest K(m) value (230.3 x 10(-4) M), followed by Streptomyces (2.17 x 10(-4) M), Cellulomonas (0.84 x 10(-4) M), and Pseudomonas (0.61 x 10(-4) M). The K(m) values and the linearity obtained from Streptomyces (2.6 mmol/L), Pseudomonas (2.1 mmol/L), or Cellulomonas (2.1 mmol/L) were too low. Dichlorophenol isomers, acting as inhibitors, increased the enzyme's K(m). The addition of 3,4-dichlorophenol raised the K(m) of Streptomyces from 2.17 x 10(-4) to 24.89 x 10(-4) M. The linearity was increased from 2.6 to 13.0 mmol/L. The high K(m) of Brevibacterium resulted in an insensitive reaction and low cholesterol linearity (7.8 mmol/L). An increase in the sample-to-reagent ratio from 1:100 to 1:10 enhanced the reaction rate and the linearity from 7.8 to 20.7 mmol/L. We suggest that Brevibacterium and Streptomyces cholesterol oxidase (with the addition of 3,4 dichlorophenol) are good sources for serum cholesterol determination by the kinetic method.