To optimize bioremediation strategies, knowledge of which bacterial groups are actually degrading specific hydrocarbon fractions is required. In this study, we monitored the utilization rate of unlabeled (12C) and labeled (13C) substrates, benzene (0.559 μL L-1 h-1) and hexadecane (0.330 μL L-1 h-1) in seawater pre-exposed to hydrocarbons over 72 h in laboratory based microcosms. Microbial community analysis by RNA-SIP DGGE showed substantial differences between the banding pattern of 12C and 13C communities. Cluster analysis of the microbial community profiles showed that the labeled bacterial population was ~25% similar to the original community in the unlabeled microcosms. This suggested that only a subset of the original bacterial community appeared to have utilized the labeled substrates. Sequence analysis of 16S rRNA gene sequences revealed the presence of known hydrocarbon degraders including Alcanivorax, Acinetobacter, Pseudomonas and Roseobacter. The presences of a number of Firmicutes in both sets of mesocosms suggest that these species were able to utilize both benzene and hexadecane. This study highlights the benefits of incorporating RNA-SIP in remediation studies to enhance the understanding of microbial communities in contaminated seawater.