Processes that enable marine phytoplankton to acquire trace metals are fundamental to our understanding of primary productivity and global carbon biogeochemical cycling. Here we show that the abundant marine cyanobacterium, Prochlorococcus strain MED4-Ax, has an absolute cobalt requirement and that zinc cannot substitute for cobalt in the growth medium, as is the case in some other phytoplankton species. When resuspended into fresh medium, uptake of cobalt into the cell occurs as free cobalt (Co2+). In contrast, cultures augmented with conditioned medium assimilated cobalt significantly faster than those in fresh medium, leading to the hypothesis that Prochlorococcus produced organic cobalt ligands in the conditioned medium. This work suggests that the availability of cobalt might influence the composition of phytoplankton assemblages in the open ocean.