Five dogs received a single 1.0 mg/kg dose of diazepam (DZ) IV. Concentrations of DZ and its major metabolite desmethyldiazepam (DMDZ) were simultaneously measured in plasma and cisternal cerebrospinal fluid (CSF) for up to 8 h after the dose by electron-capture gas-liquid chromatography. DZ was rapidly eliminated from plasma (half-life 0.3--1.3 h); DZ disappearance was mirrored by formation of DMDZ, which in turn was eliminated slowly, Both DZ and DMDZ rapidly penetrated CSF and concentrations in CSF declined parallel with those in plasma. Despite rapid uptake, the extent of CSF transfer of DZ and DMDZ was limited by plasma protein binding. Mean CSF:plasma concentrtion ratios for DZ (range 0.023--0.137) and DMDZ (range 0.047--0.119) were highly correlated with the unbound fraction in plasma (r = 0.95 and 0.80, respectively). Thus DZ and DMDZ concentrations in CSF, presumed to reflect concentrations at the site of action, are determined by unbound plasma concentrations. The intensity of pharmacologic action is more likely to correlate with unbound than with total plasma concentrations.