Centimetre-scale vertical distribution of mineralization potential was determined for 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) by 96-well microplate radiorespirometric analysis in aquifer sediment sampled just below the groundwater table. Mineralization of 2,4-D and MCPA was fastest in sediment samples taken close to the groundwater table, whereas only minor mineralization of MCPP was seen. Considerable variability was exhibited at increasing aquifer depth, more so with 2,4-D than with MCPA. This suggests that the abundance of MCPA degraders was greater than that of 2,4-D degraders, possibly due to the fact that the overlying agricultural soil had long been treated with MCPA. Mineralization of 2,4-D and MCPA was followed by increased abundance of tfdA class I and class III catabolic genes, which are known to be involved in the metabolism of phenoxy acid herbicides. tfdA class III gene copy number was approximately 100-fold greater in samples able to mineralize MCPA than in samples able to mineralize 2,4-D, suggesting that tfdA class III gene plays a greater role in the metabolism of MCPA than of 2,4-D. Degradation rate was found to correlate positively with tfdA gene copy number, as well as with the total organic carbon content of the sediment.