AIMS To determine the effect of radiation dose on biochemical control in prostate cancer patients treated in a single institution with three-dimensional conformal radiotherapy (3DCRT) and the additional effect of androgen deprivation in prostate cancer patients. MATERIALS AND METHODS In total, 363 men with T1-T3b prostate cancer treated in a sequential radiation dose-escalation trial from 66.0 to 84.1 Gy (International Commission Radiation Units and Measurement [ICRU] reference point) between 1995 and 2003, and with a minimum follow-up of 24 months, were included in the analysis. One hundred and forty-eight (41%) men were treated with 3DCRT alone; 74 (20%) men received neoadjuvant androgen deprivation (NAD) 4-6 months before and during 3DCRT; and 141 (39%) men received NAD and adjuvant androgen deprivation (AAD) 2 years after 3DCRT. Univariate, stratified and multivariate analyses were carried out separately for defined risk groups (low, intermediate and high) to determine the effect of radiation dose on biochemical control and its interaction with hormonal manipulation and clinical prognostic variables. RESULTS The median follow-up was 59 months (range 24-147 months). The actuarial biochemical disease-free survival (bDFS) at 5 years for all patients was 75% (standard error 3%). For low-risk patients, the bDFS was 82% (standard error 5%), for intermediate-risk patients it was 64% (standard error 6%) and for high-risk patients it was 77% (standard error 3%) (P = 0.031). In stratified and multivariate analyses, high-dose 3DCRT for all risk groups, and for high-risk patients, the use of long-term AAD vs NAD, contributed independently and significantly to improve the outcome of prostate cancer patients. CONCLUSION The present study indicates an independent benefit on biochemical outcome of high-dose 3DCRT for low-, intermediate- and high-risk patients and of long-term AAD in high-risk prostate cancer patients.