Estuaries are among the most productive of aquatic ecosystems. Yet the collective understanding of patterns and drivers of primary production in estuaries is incomplete, in part due to complex hydrodynamics and multiple controlling factors that vary at a range of temporal and spatial scales. A whole-ecosystem experiment was conducted in a deep, pelagically dominated terminal channel of the Sacramento-San Joaquin Delta (California, USA) that seasonally appears to become nitrogen limited, to test whether adding calcium nitrate would stimulate primary productivity or increase phytoplankton density. Production did not respond consistently to fertilization, in part because nitrate and phytoplankton were dispersed away from the manipulated area within 1–3 days. Temporal and spatial patterns of gross primary production were more strongly related to stratification and light availability (i.e., turbidity) than nitrogen, highlighting the role of hydrodynamics in regulating system production. Similarly, chlorophyll was positively related not only to stratification but also to nitrogen—with a positive interaction—suggesting stratification may trigger nutrient limitation. The average rate of primary production (4.3 g O₂ m⁻² d⁻¹), metabolic N demand (0.023 mg N L⁻¹ d⁻¹), and ambient dissolved inorganic nitrogen concentration (0.03 mg N L⁻¹) indicate that nitrogen can become limiting in time and space, especially during episodic stratification events when phytoplankton are isolated within the photic zone, or farther upstream where water clarity increases, dispersive flux decreases, and stratification is stronger and more frequent. The role of hydrodynamics in organizing habitat connectivity and regulating physical and chemical processes at multiple temporal and spatial scales is critical for determining resource availability and evaluating biogeochemical processes in estuaries.