A novel microliter volume flow through electrochemical cell incorporating a Hg-Ir microelectrode for on-line coupling to hollow fiber permeation liquid membrane (PLM) for trace metal separation and preconcentration probe to determine metal speciation in natural water is described. The permeation liquid membrane contained 0.1 mol L−1 didecyl diaza-crown-ether and 0.1 mol L−1 lauric acid dissolved in toluene/phenylhexane mixture. The membrane acts as a barrier between the sample solution and a strip solution containing a complexant into which the extracted metals are back extracted. This membrane transports selectively the trace metal ions that are capable of binding to the carrier, in particular Cu, Pb, Cd and Zn. High preconcentration factors are obtained using small volumes of strip solution and large sample volume. Combination of this chemical sensor to voltammetric detectors allows one to determine free metal concentration in real time and eases automation. The difficulties linked with the use of a microliter voltammetric cell for the application sought, the optimal cell design as well as optimal conditions for permitting on-line determination of Pb and Cu are described. The advantage of using PLM-ASV coupling with respect to ASV for the determination of trace metals in natural waters are that matrix effects, which are often a problem in ASV, are minimized and the sensitivity of the method is increased because of the additional preconcentration from the PLM prior to ASV determination. After testing the system with a synthetic solution, applicability for speciation of lead and copper ion in river waters were assessed. Comparison of the results with off-line metal determination by graphite furnace atomic absorption spectrometry has also been reported. Preconcentration factors of ca. 800 and ca. 300 in 20 minutes for Pb and Cu, respectively, were obtained using 50 μL strip and 250 mL source solutions. Under the working conditions used, e.g., with 20 minutes PLM preconcentration time, detection limits of ca. 0.02 nmol L−1 can be achieved.