A combined culture and field study was conducted in order to 1) more firmly identify the physiological controls on hydrogen isotopic composition of C37 alkenones produced by open-ocean coccolithophores and 2) determine the degree to which these controls are manifested in a natural water column. Nutrient-limitation experiments in culture, combined with previously published data, show that net fractionation between the growth medium and alkenones (αK37) varies with cellular alkenone content and production rate, and, by extension, growth phase. It is hypothesized that the relationship of αK37 with cellular alkenone content and production rate is due to increased use of anabolic NADPH in response to high rates of lipid synthesis. Euphotic zone profiles of δDK37, measured in suspended material from the Gulf of California and Eastern Tropical North Pacific, decreased with depth and light availability, and did not correlate in any expected way with previously-suggested controls on αK37. It is possible that the field data are driven by behavior in light-limited cells that is not represented by the available, nutrient-limited culture data. If true, δDK37 may have utility as an indicator of production depth in settings prone to subsurface production maxima. Relationships between αK37, cell density, and the carbon-isotopic fractionation term εp, however, suggest that αK37 acts an indicator of growth rate, which in this setting is only partially dependent on light, consistent with our interpretation of the culture data. If this latter interpretation proves correct, δDK37 may be a powerful ecological proxy specific to these climatically-important, calcifying, temperature-encoding species.