Direct evidence for metabolism in a variety of frozen environments has pushed temperature limits for bacterial activity to increasingly lower temperatures, so far to −20 °C. To date, the metabolic activities of marine psychrophilic bacteria, important components of sea-ice communities, have not been studied in laboratory culture, not in ice and not below −12 °C. We measured [3H]-leucine incorporation into macromolecules (further fractionated biochemically) by the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H over a range of anticipated activity-permissive temperatures, from +13 to −20 °C, including expected negative controls at −80 and −196 °C. For incubation temperatures below −1 °C, the cell suspensions [all in artificial seawater (ASW)] were first quick-frozen in liquid nitrogen. We also examined the effect of added extracellular polymeric substances (EPS) on [3H]-leucine incorporation. Results showed that live cells of strain 34H incorporated substantial amounts of [3H]-leucine into TCA-precipitable material (primarily protein) down to −20 °C. At temperatures from −1 to −20 °C, rates were enhanced by EPS. No activity was detected in the killed controls for strain 34H (or in Escherichia coli controls), which included TCA-killed, heat-killed, and sodium azide- and chloramphenicol-treated samples. Surprisingly, evidence for low but significant rates of intracellular incorporation of [3H]-leucine into protein was observed for both ASW-only and EPS-amended (and live only) samples incubated at −80 and −196 °C. Mechanisms that could explain the latter results require further study, but the process of vitrification promoted by rapid freezing and the presence of salts and organic polymers may be relevant. Overall, distinguishing between intracellular and extracellular aspects of bacterial activity appears important to understanding behavior at sub-freezing temperatures.