Measurements of neuropeptides, neurotransmitters and amino acids in freely moving rat brain were performed by microdialysis method. Recently, despite the different methods of measurement for each of those compounds, there have been attempts to measure them simultaneously from the same sample of brain dialysate, without measuring each compound from different samples. However, more sophisticated techniques and highly sensitive devices are required, to manage smaller volume samples obtained with this recent method. In our study, we present W-probe consisting of two probes with cylindrical dialysis membrane with 3 mm length and 0.222 mm outer diameter, intending to improve this method of analysis, without decreasing the recovery rate and sample volume compared with U-probe and permitting a clear analysis of acetylcholine and monoamine metabolites, even with the use of eserine. The results demonstrated that sample volume did not decrease even with slow perfusion rate (2 microliters/min). Thus, the sampling interval could be shorter than the previous microdialysis method, permitting the measurement of neurotransmitter releasing variation.