The pool of cellular chelatable iron ('free iron', 'low-molecular-weight iron', the 'labile iron pool') is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8+/-2.6 microM within the cytosol and of at least 4.8 microM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6+/-2.9 microM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3+/-2.6 microM; nucleus, 11.8+/-3.9 microM; mitochondria, 9.2+/-2.7 microM); in addition, chelatable iron (approx. 16+/-4 microM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.