The importance of urea in supplying the nitrogen (N) required by planktonic communities has long been recognized, notably by James J. McCarthy in studies as early as the 1970s. Utilization of urea involves a two-step enzymatic process in phytoplankton, with urea first entering the cell via transport (i.e., urea uptake), followed by the conversion of urea into ammonium by the enzyme urease. This article describes a series of field observations and experiments conducted in the Chesapeake Bay, USA, from 2001 through 2018, aimed at understanding the relationship between urea uptake and urease activity and the role of environmental factors on that relationship. Principal component analysis revealed a few patterns. Urea uptake, for example, was consistently positively related to combined variables that included urea concentrations. Similarly, urease activity was consistently positively related to combined variables that included temperature. Contrary to findings in culture studies, however, relationships with environmental factors within different phytoplankton taxa in the field were not clear. This suggests that factors other than those examined may be involved in the regulation of urea uptake and urease activity. New insights into the role of the urea cycle in phytoplankton nitrogen dynamics suggest that the regulation of urease may not be directly impacted by environmental factors, but indirectly regulated by different metabolic pathways responding to nutrient availability, light, and temperature conditions.