The variety of kinetics expressions encountered in the literature and the unreasonably broad range of values reported for the kinetics constants of Acidithiobacillus ferrooxidans underscore the need for a unifying experimental procedure and for the development of a reliable kinetics equation. Following an extensive and critical review of reported experimental techniques, a method based on batch pH-controlled kinetics experiments lasting less than one doubling time was developed for the determination of extant kinetics constants. The Fe(II) concentration in the experiments was measured by a method insensitive to Fe(III) interference. Kinetics parameters were determined by nonlinear fitting of the integrated form of the Monod equation to yield a KS of 31 ± 4 mg Fe2+ liter-1 (mean ± standard deviation), a KP of 139 ± 20 mg Fe3+ liter -1, and a μmax of 0.082 ± 0.002 h-1. The corresponding kinetics equation was as follows: dS/dT=(-0.082/2. 3·107)S·X/31(1+P0+S0-S/139)+S where S represents the Fe(II) concentration in mg liter-1, P 0 represents the initial Fe(III) concentration in mg liter -1, X represents the suspended bacterial cell concentration in cells ml-1, and t represents time in hours. The measured data fit this equation exceptionally well, with an R2 of >0.99. Fe(III) inhibition was found to be of a competitive nature. Contrary to previous reports, the results show that the concentration of Acidithiobacillus ferrooxidans cells has no affect on the kinetics constants. The kinetics equation can be considered applicable only to A. ferrooxidans cells grown under environmental conditions similar to those of the inoculum tested in the study. In contrast, the experimental and computational procedure is completely general and can be applied to A. ferrooxidans irrespective of the culture history.