AbstractMicrospectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6‐phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation‐reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular inter‐conversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two‐step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two‐step NAD(P)H response to sequentially injected fructose 1,6‐diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell‐to‐cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.