Differential fractionation of inorganic carbon stable isotopes during photosynthesis is an important cause of variability in algal carbon isotope signatures. Several physiological models have been proposed to explain algal photosynthetic fractionation factors (εp). These models generally consider CO2 concentration, growth rate, or cell morphometry and have been supported by empirical evidence from laboratory cultures. Here, we explore the applicability of these models to a broad range of lakes with mixed phytoplankton communities. Understanding this fractionation is necessary for using carbon stable isotopes for studies ranging from food webs to paleolimnology. In our largest comparative study, values of δ13C-POC ranged from –35.1 ‰ to –21.3 ‰. Using several methods to obtain an algal isotopic signature, we found high variability in fractionation among lakes. There was no relationship between εp and one of the most important predictors in existing models, pCO2. A whole-lake inorganic 13C addition was used to create distinct algal isotope signatures to aid in examining εp. Measurements and a statistical model from the isotope addition revealed that algal fractionation was often low (0 – 15 ‰).