The fluorescence concentration relationship of formaldehyde-induced fluorescence of noradrenaline in adrenergic nerves of rat iris was investigated by the use of isotope and microfluorimetric techniques. The irides first were depleted of their endogenous stores of noradrenaline either by reserpine or by the methylester of α-methyl- p-tyrosine (H 44/68) and then were incubated in vitro in a physiologic medium containing 3H-noradrenaline for partial replenishment of the nerves of their transmitter. The incubated irides were prepared as whole amounts and were exposed to gaseous formaldehyde for histochemical demonstration of noradrenaline. Microfluorimetric and isotope measurements were performed on the same preparations. The fluorescence intensity was found to be proportional to the noradrenaline concentration up to a value corresponding to 30-40% of the endogenous level, above which a concentration-dependent quenching of the fluorescence occurred. In the linear part of the relation, it was possible to perform fairly safe estimations of the fluorescence intensity by eye, but, if the nerves contained more than 40% of the endogenous content, usually no differentiation could be made, probably as a result of quenching effects. The quenching appears somewhat earlier and the relative fluorescence yield is lower when noradrenaline is stored in the intraneuronal amine storage granules, compared with the situation when the amine mainly is distributed extragranularly in the axoplasm. It can be concluded that, if changes in fluorescence intensity as compared with the control are observed, this reflects true changes in amine concentrations, but changes in amine concentration may escape detection when the concentration of amine is so high that quenching occurs.