SUMMARY.1. The relationship between microbial ATP and biomass‐ carbon in the near surface waters (0.5–5 m) of eight New Zealand lakes was studied to determine the constancy of the carbon:ATP ratio under natural growth conditions. Concentrations of microbial carbon were estimated indirectly from cell volume determinations.2. The carbon:ATP ratio remained reasonably constant (interquartile range 248–291,n= 50), except during periods of nitrogen/phosphorus deficiency when carbon: ATP ratios increased to values greater than 400.3. During periods of nitrogen/phosphorus sufficiency, corresponding estimates of microbial ATP and biomass‐carbon were strongly correlated (r=0.97,n=47) and related by the equation carbon = (287±20) ATP‐(22±41) where carbon and ATP are expressed in mg m−3. From this relationship an average carbon:ATP ratio of 267 (SE=5) was calculated.4. This ratio was not significantly affected by the relative proportions of bacterial and algal biomass in the surface water samples. However, because of the marked deviation of the carbon:ATP ratio during periods of nitrogen/phosphorus deficiency, the routine use of ATP as a biomass indicator i