Failure to control localized prostate cancer can result not only in localized disease progression but also distant metastatic spread. Whereas significant advances in both surgical technique and radiation therapy have improved local control rates with decreased morbidity, consistent long-term control remains elusive. This study investigates the potential of 17-N-allylamino-17-demethoxy geldanamycin (17AAG), a geldanamycin derivative, to sensitize tumor cells to ionizing radiation, permitting a significant improvement to targeted radiotherapies of prostate carcinoma. As a monotherapeutic, 17AAG functions to modulate the action of heat shock protein 90, ultimately affecting a multitude of cellular signaling pathways. It is in Phase I trial and has shown promise in controlling prostate cancer progression. Human prostate tumor cells (LNCaP and CWR22Rv1) were grown as spheroids and incubated for 96 h with increasing doses of 17AAG immediately before and after 2 or 6 Gy low linear energy transfer (LET), high dose-rate irradiation (Cs-137 irradiator). Twelve or 24 spheroids (initial diameter, 150–200 μm) were used per experiment. Response was determined by spheroid volume measurements taken over at least 40 days, after treatment. Incubation of either cell line with 17AAG (≤1000 nm) or irradiation (≤6 Gy) alone resulted in transient median growth delays ranging from 2 to 9 days (relative to controls). Combining treatments produced dose- and cell line-dependent supra-additive responses. For LNCaP spheroids, the combination of 2 Gy and 100 nm 17AAG resulted in growth delays additive of the treatments individually; however, increasing either the radiation to 6 Gy or the 17AAG concentration to 1000 nm led to synergistic interactions. Similarly, synergy was noted in CWR22Rv1 studies at only 6 Gy and 1000 nm 17AAG. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and Ki67 staining of spheroid sections revealed the increased growth control to be a function of spheroids failing to re-enter the cell cycle. For all 6 Gy experiments, cells remaining from each of the spheroids that failed to regrow were transferred to adherent dishes to evaluate clonogenicity; growth-controlled spheroids also failed to form colonies within 2 weeks of being plated. These results suggest that significant gains in treatment effectiveness may be obtained by combining these treatment modalities, warranting additional preclinical investigation.