Tumors from 40 patients and 7 established human xenograft tumor lines were grown in three-dimensional histoculture. A Viable-Cell-Index (VCI) based on fluorescent dyes and Growth Fraction Index (GFI) based on [3H]thymidine incorporation were measured by confocal microscopy and histological autoradiography, respectively, after treatment with cytotoxic agents. Chemosensitivity in vitro with the two methods was correlated with chemosensitivity of the same set of human xenografted tumor lines grown in nude mice. The percent accuracy of in vitro to in vivo correlation with VCI (73%) was higher than GFI (63%). The number of false positives with VCI was 12.1% (4/33), and with GFI was 31.3% (10/32). The results thus indicated that in vitro histoculture with fluorescent vital-dye end-points to measure cell viability is of potential use to determine tumor chemosensitivity.