B vitamins are some of the most commonly required biochemical cofactors in living systems. Therefore, cellular metabolism of marine vitamin-requiring (auxotrophic) phytoplankton and bacteria would likely be significantly compromised if B vitamins (thiamin B ₁ , riboflavin B ₂ , pyridoxine B ₆ , biotin B ₇ , and cobalamin B ₁₂ ) were unavailable. However, the factors controlling the synthesis, ambient concentrations, and uptake of these key organic compounds in the marine environment are still not well understood. Here, we report vertical distributions of five B vitamins (and the amino acid methionine) measured simultaneously along a latitudinal gradient through the contrasting oceanographic regimes of the southern California-Baja California coast in the Northeast Pacific margin. Although vitamin concentrations ranged from below the detection limits of our technique to 30 pM for B ₂ and B ₁₂ and to ∼500 pM for B ₁ , B ₆ , and B ₇ , each vitamin showed a different geographical and depth distribution. Vitamin concentrations were independent of each other and of inorganic nutrient levels, enriched primarily in the upper mesopelagic zone (depth of 100–300 m), and associated with water mass origin. Moreover, vitamin levels were below our detection limits (ranging from ≤0.18 pM for B ₁₂ to ≤0.81 pM for B ₁ ) in extensive areas (100s of kilometers) of the coastal ocean, and thus may exert important constraints on the taxonomic composition of phytoplankton communities, and potentially also on rates of primary production and carbon sequestration.