RATIONALE AND OBJECTIVES: Although segmentation algorithms for cerebrospinal fluid (CSF), white matter (WM), and gray matter (GM) on unenhanced computed tomographic (CT) images exist, there is no complete research in this area. To take into account poor image contrast and intensity variability on CT scans, the aim of this study was to derive and validate a novel, automatic, adaptive, and robust algorithm. MATERIALS AND METHODS: Unenhanced CT scans of normal subjects from two different centers were used. The algorithm developed uses adaptive thresholding, connectivity, and domain knowledge and is based on heuristics on the shape of CT histogram. The slope of the intensity histogram corresponding to the three-dimensional largest connected region in a variable CSF intensity range is tracked to determine the critical intensity, which serves as an initial classifier of CSF-WM. Thresholds of CSF, WM, and GM are then optimally derived to minimize classification overlap. Multiple, null, and erroneous classifications are resolved by applying domain knowledge. RESULTS: The ground-truth regions with the minimal partial volume effect were used to evaluate segmentation results using the statistical markers. Average sensitivity, Dice index, and specificity, respectively, for the first center were 95.7%, 97.0%, and 98.6% for CSF; 96.1%, 97.3%, and 98.8% for WM; and 95.2%, 94.3%, and 92.8% for GM. The results were consistent for the second data center. CONCLUSIONS: The algorithm automatically identifies CSF, WM, and GM on unenhanced CT images with high accuracy, is robust to data from different scanners, does not require any parameter setting, and takes about 5 minutes in MATLAB to process a 512 × 512 × 30 scan. The algorithm has potential use in research and clinical applications.