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The chemiluminescence (CL) system K2MnO4-luminol is shown to be useful for the determination of lead(II). The method is based on the catalytic effect of Pb(II) on the CL reaction. The linear range was 3 × 10−3–9 × 10−1 mg L−1 (r = 0.9971) and the relative standard deviation (R.S.D.) for 5 × 10−2 mg L−1 Pb(II) measurement was 0.7% (n = 11). The detection limit was 3 × 10−4 mg L−1 (3σ) Pb(II). Based on this, an in vivo, on-line, real-time analytical system for monitoring the metabolism of free lead(II) in rabbit blood was developed. A microdialysis probe, implanted in the vein of a rabbit, was perfused with perfusate at a flow rate of 5 µL min−1. The concentration of free lead(II) in the dialysate was determined on-line with a flow-injection CL system. This system included microdialysis sampling, on-line separation and chemiluminescence detection. The concentration-time curve of lead(II) was in accordance with the one-compartmental open model, T1/2 (elimination half-life), Tmax (peak time) and Cmax (peak concentration) were 37.77 min, 85.20 min and 0.137 mg L−1, respectively.