The biocatalytic potential of the NADH-dependent p-hydroxybenzoate hydroxylases (PHBH) from Rhodococcus rhodnii 135 and Rhodococcus opacus 557 was investigated. Monofluorinated 4-hydroxybenzoates were efficiently hydroxylated, albeit at different rates. 2-Fluoro-4-hydroxybenzoate was a true substrate for PHBH from R. rhodnii 135 but a substrate inhibitor for PHBH from R. opacus 557. Monochlorinated 4-hydroxybenzoates also acted as PHBH substrates, but with these compounds strong uncoupling of hydroxylation (formation of hydrogen peroxide) occurred. PHBH from R. rhodnii 135 preferred the 5'-hydroxylation of 2-chloro-4-hydroxybenzoate but the enzyme from R. opacus 557 favored the formation of 2-chloro-3,4-dihydroxybenzoate. Conversely, PHBH from R. rhodnii 135 regioselectively hydroxylated 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate whereas the enzyme from R. opacus 557 also produced significant amounts of 2-fluoro-4,5-dihydroxybenzoate. At high NADH/substrate ratio, both 2-fluorodihydroxybenzoate products were further converted to 2-fluoro3,4,5-trihydroxybenzoate. PHBH from R. rhodnii 135 and R. opacus 557 preferred the 5'-hydroxylation of 3-chloro-4-hydroxybenzoate. However, conversion of 3-fluoro-4-hydroxybenzoate involved considerable dehalogenation affording nearly equal amounts of 3,4-dihydroxybenzoate and 5-fluoro-3,4-dihydroxybenzoate. At high NADH/substrate ratio, the latter compound was further converted to 3,4,5-trihydroxybenzoate. The results are discussed in relation to the properties of the NADPH-specific PHBH from Pseudomonas fluorescens.