Although the biochemical functions and biosynthetic pathways of alkenones are still largely unknown, alkenone unsaturation ratios are now used extensively to infer ancient sea surface temperature, and their isotopic compositions have been used to reconstruct ancient atmospheric CO2 levels. The inferred relationships between alkenone unsaturation ratios, isotopic compositions, and growth conditions are based on empirical laboratory and field studies and, in the case of isotope fractionation, on simple models of carbon acquisition and fixation. Significant uncertainty still exists concerning the physiological and ecological factors affecting cellular production of alkenones, unsaturation ratios, and isotopic composition. Phytoplankton culture conditions have been shown to affect alkenone unsaturation (U37K′), cellular alkenone content, intracellular isotopic compositions (Δδ), and changes in fractionation (εP) as a function of the quotient of algal growth rate and aqueous carbon dioxide concentration (μ/CO2). Such studies imply that plant physiology can affect the interpretation of environmental signals. The factor(s) controlling cellular alkenone concentrations and unsaturation ratios are reviewed, as well as the carbon isotopic composition of the alkenone-producing algae. A new technique is presented to determine growth rates of the alkenone-containing algae in natural settings that will facilitate testing laboratory-based hypotheses concerning the carbon isotopic fractionation and its relationship to growth rate/growth status of alkenone-producing algae in the field.