Pentachloronitrobenzene (PCNB) is an organochlorine fungicide used either as seed dressing or for soil treatment. Research was conducted to investigate the microbial reductive transformation of PCNB with cultures developed from a contaminated estuarine sediment. The biotransformation of PCNB to pentachloroaniline (PCA) occurred under all electron accepting conditions tested. Sequential dechlorination of PCA to di- and in some cases to mono-chlorinated anilines occurred under fermentative/methanogenic conditions. Based on the use of inhibitors, methanogens were not involved in the sequential dechlorination of PCA. Based on 16S rRNA gene analysis, among five known dechlorinating bacterial groups tested, only Dehalococcoides was detected in the mixed culture. The sequential dechlorination of PCA was simulated using a branched-chain Michaelis-Menten kinetic model. The dechlorination rate (k) of the chlorinated anilines ranged from 0.25 to 1.19 uM/day and the half-saturation coefficient (KC) ranged from 0.11 to 1.72 uM at an incubation temperature of 22C and pH 6.90.1. Incubation at different temperature and pH values resulted in significant differences in the biotransformation rate and extent of PCNB in the fermentative/methanogenic enrichment culture. Incubation at 35C resulted in significantly different product distribution. The effect of temperature on the PCA dechlorination rate was modeled using an Arrhenius relationship. Dechlorination of PCA and methanogenesis were not observed in cultures amended with completely bioavailable iron sources until all Fe3+ was reduced to Fe2+. In contrast, PCA dechlorination took place at the same time with iron reduction in the same mixed, methanogenic culture amended with a less bioavailable iron source (FeOOH). PCA was sequentially dechlorinated to dichloroanilines in cultures amended with low nitrate concentrations, whereas partial dechlorination of PCA to tetrachloroanilines was observed in cultures amended with high initial nitrate concentrations due to the accumulation of reduced nitrogen species (e.g., NO, N2O). A semi-empirical molecular model (MOPAC/AM1) was used to estimate the thermodynamic and electronic properties of all chlorinated aniline congeners. These values were used to predict the sequential PCA dechlorination pathway and compare to experimentally observed dechlorination reactions. The results of this study have significant environmental implications relative to the fate and transport of PCNB, PCA and its dechlorination products in subsurface systems.