Cobalamin influences marine microbial communities because an exogenous source is required by most eukaryotic phytoplankton, and demand can exceed supply. Pseudocobalamin, psB12, is a cobalamin analog that is produced and used by most cyanobacteria but is not directly available to eukaryotic phytoplankton. A subset of microbes can remodel pseudocobalamin into cobalamin, but a scarcity of pseudocobalamin measurements impedes our ability to evaluate its importance for marine cobalamin production. Here, we perform simultaneous measurements of pseudocobalamin and methionine synthase (MetH), the key protein that uses it as a co-factor, inSynechococcuscultures and natural communities. InSynechococcussp. WH 8102, pseudocobalamin quota decreases in low temperature (17 °C) and low N:P, while MetH did not. Pseudocobalamin and MetH quotas were influenced by culture methods (batch vs semi-continuous) and growth phase (exponential vs stationary). Despite the variability present in cultures, we found a comparably consistent quota of 296 ± 101 pseudocobalamin molecules per cyanobacterial cell in the Northwest Atlantic Ocean, suggesting that cyanobacterial cell counts may be sufficient to estimate pseudocobalamin inventories in this region. This work offers insights into cellular pseudocobalamin metabolism and the environmental and physiological conditions that may influence it and provides environmental measurements to further our understanding of when and how pseudocobalamin can influence marine microbial communities.