The survival and effectiveness of a bioaugmentation strain in its target environment depend not only on physicochemical parameters in the soil but also on the physiological state of the inoculated organism. This study examined the effect of variations in inoculum pretreatment on the survival, metabolic activity (measured as rRNA content) and polycyclic aromatic hydrocarbon (PAH)-catabolic gene expression of Sphingobium yanoikuyae B1 in an aged PAH-contaminated soil. RNA denaturing gradient gel electrophoresis analysis showed stable colonization of PAH-contaminated soil by S. yanoikuyae B1 after four pretreatments (growth in complex or minimal medium, starvation, or acclimation to phenanthrene). By contrast, extractable CFUs decreased with time for all four treatments, and significantly faster for Luria Bertani-grown inocula, suggesting that these cells adhered strongly to soil particles while remaining metabolically active. Pretreatment of the inoculum had a dramatic effect on the expression of genes specific to the PAH-degradation pathway. The highest levels of bphC and xylE expression were seen for inocula that had been precultivated on complex medium, and degradation of PAHs was significantly enhanced in soils treated with these inocula. The results suggest that using complex media instead of minimal media for cultivating bioaugmentation inocula may improve the subsequent efficiency of contaminant biodegradation in the soil.