The influence of phosphate-free medium buffered with synthetic organic buffers, and of a preliminary incubation of cells in medium lacking added substrate ('pre-incubation') was investigated with mouse-cultured Ehrlich ascites tumour cells. In comparison to phosphate-containing bicarbonate-buffered balanced-salts medium, organic-buffered medium, without a preliminary substrate-free pre-incubation, was associated with 20-30% reduction in the rate of glycolysis, the 3- to 4-fold accumulation of fructose 1,6-bisphosphate and the halving of both ATP and total adenine nucleotide levels. These perturbations were reversed by the inclusion of 5 mM sodium phosphate in the organic-buffered medium. Pre-incubation for up to 90 min, before inclusion of glucose, resulted in greater depression of the glycolytic rate and concentrations of adenine nucleotides. This occurred in both the balanced-salts medium and the organic-buffered medium. During pre-incubation cells were lysed, releasing lactate dehydrogenase, when physically agitated too vigorously. It was concluded that the use of phosphate-free medium and pre-incubation are not advisable procedures for routine metabolic investigations with this cell line.