Abstract: The present study compared two different in vivo microdialysis methods which estimate the extracellular concentration of analytes at a steady state where there is no effect of probe sampling efficiency. Each method was used to estimate the basal extracellular concentration of dopamine (DA) in the nucleus accumbens of the rat. In the first method, DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 2.5, 5, and 10 nM) and DA is measured in the dialysate from the brain to generate a series of points which are interpolated to determine the concentration of no net flux. Using this method, basal DA was estimated to be 4.2 ± 0.2 nM (mean ± SEM, n = 5). The slope of the regression gives the in vivo recovery of DA, which was 65 ± 5%. This method was also used to estimate a basal extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) concentration in the nucleus accumberis of 5.7 ± 0.6 μM, with an in vivo recovery of 52 ± 11% (n = 5). A further experiment which extended the perfusate concentration range showed that the in vivo recovery of DA is significantly higher than the in vivo recovery of DOPAC (p < 0.001), whereas the in vitro recoveries of DA and DOPAC are not significantly different from each other. The in vivo difference is thought to be caused by active processes associated with the DA nerve terminal, principally release and uptake of DA, which may alter the concentration gradient in the tissue surrounding the probe. The second method measures dialysate DA at several perfusion flow rates (0.1, 0.2, 0.4, and 1.2 μ1/min) and extrapolates the data to zero flow using a nonlinear least squares regression. This method estimated a basal extracellular DA concentration of 3.9 ± 0.2 nM (n = 5). The two independent methods are in reasonable agreement that the extracellular concentration of DA in the nucleus accumbens is about 4 nM.