This study describes an investigation of the factors regulating spatial and temporal variability of bacterioplankton carbon metabolism in aquatic ecosystems using the tidal creeks of a temperate salt-marsh estuary as a study site. Differences in land-use and landscape characteristics in the study site (Monie Bay) generate strong predictable gradients in environmental conditions among and within the tidal creeks, including salinity, nutrients, and the quality and quantity of dissolved organic matter (DOM). A 2-yr study of bacterioplankton metabolism in this system revealed a general positive response to system-level nutrient enrichment, although this response varied dramatically when tidal creeks differing in salinity were compared. Of the numerous environmental parameters investigated, temperature and organic matter quality had the greatest influence on carbon metabolism. All measures of carbon consumption (i.e., bacterioplankton production (BP), respiration (BR) and total carbon consumption (BCC)) exhibited significant positive temperature dependence, but the disproportionate effect of temperature on BP and BR resulted in the negative temperature dependence of bacterioplankton growth efficiency (BGE = BP/[BP+BR]). Dissolved organic matter also had an influence on carbon metabolism, with higher BCC and BGE generally associated with DOM of greater lability. Our exploration of factors driving this pattern suggests that the energetic content and lability of DOM may be more important than nutrient content or dissolved nutrients alone in determining the magnitude and variability of BGE. Investigations of single-cell activity revealed that BCC and BGE may be further modulated by the abundance, proportion, and activity of highly-active cells. Differences in single-cell activity among creeks differing in freshwater input also imply that other cellular-level properties (e.g., phylogenetic composition) may be an important factor. Collectively, results from this research indicate that the variability of bacterioplankton carbon metabolism in temperate estuarine systems represents a complex response to a wide range of environmental and biological factors, of which temperature and DOM quality appear to be the most important. Furthermore, this research reveals fundamental differences in both cellular and community-level metabolic processes when freshwater and marine endmembers of estuaries are compared that may contribute to the variability in bacterioplankton carbon metabolism within and among estuarine systems.