Monochlorobimane (MCB) has been used as a glutathione (GSH) specific fluorescent probe capable of delineating GSH heterogeneity in cellular systems. Generally, low concentrations of MCB (<50 µm) have been used to quantitatively label GSH in rodent cell lines. Incubation of the hamster cell lines, CHO AB1 and V79, with 10 µm MCB labeled 75 and 39% of the reduced GSH pool, respectively. In contrast, incubation of 7 different human cell lines with 10 µm MCB labeled less than 4% of the total reduced GSH pool. The human cell lines required 1000 µm MCB to label an average of 73% of the GSH pool (range, 60–88%). When using 1000 µm MCB to label GSH, flow cytometry results from 7 different cell lines (human and rodent) were in good agreement with high performance liquid chromatography and standard spectrophotometric analysis with regards to a rank ordering of the GSH content determined for each cell line. The human glutathione S-transferases B2B2, B1B2, ψ, π, and the rat transferases 1–2, 3–3, and 3–4 were isolated and purified for steady state kinetic analysis with MCB and GSH as the primary substrates. The human basic transferases, B1B2 and B2B2, had Km values for MCB of 354 and 283 µm and Vmax values of 33.3 and 34.6 µmol bimane-GSH/min/mg protein, respectively. The rat basic transferase 1–2 showed similar kinetic results with a Km of 199 µm and a Vmax of 35.5 µmol bimane-GSH/min/mg protein. The human neutral transferase (ψ) had a Km for MCB of 204 µm with a Vmax of 6.5 µmol bimane-GSH/min/mg protein. In contrast, MCB has a high affinity for the rat neutral transferase with a Km of 2.6 µm and a Vmax of 35.1 µmol bimane-GSH/min/mg protein. The human acidic transferase (π), the predominate transferase found in most human tumor cell lines, has a Km of 264 µm for MCB and a Vmax of 1.99 µmol bimane-GSH/min/mg protein. The kcat/Km values indicated that MCB is an excellent substrate for the rat neutral transferases while the human π glutathione S-transferase showed the least reactivity. Collectively the data indicate that MCB fails to label GSH at lower concentrations (<50 µm) in human cell lines because of the reduced affinity of MCB for the human transferases and possibly also due to differences in glutathione S-transferase isozyme expression between rodent and human cell lines.