Didanosine is commonly prescribed as monotherapy or as part of a combination regimen for patients with human immunodeficiency virus infection. The use of lower doses, either as part of a combination regimen or as a result of dose reduction secondary to clinical intolerance, requires that a sensitive assay method be available for either traditional or population-based pharmacokinetic evaluations. We evaluated a radioimmunoassay technique with a standard curve range of 0 to 100 ng/ml in human plasma, urine, and cerebrospinal fluid and assessed its accuracy and precision for use in pharmacokinetic studies.