A classic equation that has frequently been used to estimate the fraction of glucose metabolized by the pentose phosphate pathway, using 14CO2 data, is more simply re-derived with careful consideration of the assumptions involved and the conditions under which it is applicable. The equation is shown to be unreliable for non-homogeneous tissues, depending on the fraction of triose phosphate converted to CO2. The formula in question is as follows: ([1]CO2/G-[6]CO2/G)/(1-[6]CO2/G) = 3Fmet./(1 + 2Fmet.) where [1]CO2 and [6]CO2 are output rates of carbons 1 and 6 of glucose respectively to CO2, G is the rate of glucose uptake and Fmet. is the fraction of the glucose that is metabolized to CO2 and triose phosphate by the pentose phosphate pathway, allowing for recycling of an appropriate fraction of the fructose-6-phosphate produced by the pathway. This analysis illustrates the importance of suitably testing any equation that assumes homogeneity before application to non-homogeneous tissues.