SUMMARY: A facultatively oligotrophic ultramicrobacterium (strain RB2256) isolated from an Alaskan fjord by extinction dilution in seawater, was grown in batch culture and under single- and dual-substrate-limitation of alanine and glucose in a chemostat. The nature of the uptake systems, and the uptake kinetics and utilization patterns of alanine and glucose were investigated. Glucose uptake was inducible, the system exhibited a narrow substrate specificity, and part of the uptake system was osmotic-shock-sensitive. Half-saturation constants for glucose were between 7 and 74 μM during glucose limitation. The initial step in glucose metabolism was the synthesis of sugar polymers, even during glucose-limited growth. The alanine uptake system was constitutively expressed and was binding-protein-dependent. In addition to L-alanine, nine other amino acids inhibited accumulation of [14C]L-alanine, indicating broad substrate specificity of the alanine transporter. Half-saturation constants between 1-3 and 1-8 μM were determined for alanine uptake during alanine limitation. Simultaneous utilization of glucose and alanine occurred during substrate-limited growth in the chemostat, and during growth in batch culture at relatively high (mM) substrate concentrations. However, the half-saturation constant for alanine transport during dual-substrate-limitation, i.e. in the presence of glucose, increased almost fivefold. We conclude that mixed substrate utilization is an inherent property of this organism.