The relationship between the nitrogen and carbon isotopic ratios of chlorophyll a and total biomass was explored in cultured marine phytoplankton to assess the utility of chlorophyll as an isotopic proxy for photoautotrophs. A near constant nitrogen isotopic depletion of 5.06 +/- 1.13%% (95% confidence interval) in chlorophyll a relative to total nitrogen was observed in 8 species. This value was similar to isotopic differences between chlorophyll a and marine particles (5.27 +/- 1.48%% (1σ); n = 6) and sediments (5.39 +/- 0.67%% (1σ); n = 4) in a variety of settings. These findings suggest that a 5.1%% isotopic depletion of chlorophyll a relative to total algal nitrogen is a robust relationship that justifies the use of chlorophyll as a nitrogen isotopic surrogate for photoautotrophs. Although interspecies differences in Δδ 1 5 N c e l l - C h l a exist, and growth rate has a small effect on this parameter, the field data suggest these factors are probably minimized in the ocean where multiple species and growth rates occur. The nitrogen isotopic depletion of chlorophyll a probably occurs during the transamination of glutamic acid in δ-aminolevulinic acid biosynthesis, the first committed precursor to chlorophyll.The carbon isotopic composition of chlorophyll from 12 batch cultures and 7 species of marine phytoplankton was elevated by 0.32 +/- 1.61%% (95% confidence interval) relative to total cellular carbon. No significant interspecies variance was observed that was not attributable to intraspecies variance. There was a moderate inverse correlation between growth rate and Δδ 1 3 C c e l l - C h l a , and it is hypothesized that this parameter is largely responsible for the large range of intraspecies Δδ 1 3 C c e l l - C h l a values observed in batch cultures.