Androgen‐ablation is a most commonly prescribed treatment for metastatic prostate cancer but it is not curative. Development of new strategies for treatment of prostate cancer is limited partly by a lack of full understanding of the mechanism by which androgen regulates prostate cancer cell proliferation. This is due, mainly, to the limitations in currently available experimental models to distinguish androgen/androgen receptor (AR)‐induced events specific to proliferation from those that are required for cell viability. We have, therefore, developed an experimental model system in which both androgen‐sensitive (LNCaP) and androgen‐independent (DU145) prostate cancer cells can be reversibly blocked in G₀/G₁ phase of cell cycle by isoleucine deprivation without affecting their viability. Pulse‐labeling studies with ³H‐thymidine indicated that isoleucine‐deprivation caused LNCaP and DU145 cells to arrest at a point in G₁ phase which is 12–15 and 6–8 h, respectively, before the start of S phase and that their progression into S phase was dependent on serum factors. Furthermore, LNCaP, but not DU145, cells required AR activity for progression from G₁ into S phase. Western blot analysis of the cell extracts prepared at regular intervals following release from isoleucine‐block revealed remarkable differences in the expression of cyclin E, p21^Cip1, p27^Kip1, and Rb at the protein level between LNCaP and DU145 cells during progression from G₁ into S phase. However, in both cell types Cdk‐2 activity associated with cyclin E and cyclin A showed an increase only when the cells transited from G₁ into S phase. These observations were further corroborated by studies using exponentially growing cells that were enriched in specific phases of the cell cycle by centrifugal elutriation. These studies demonstrate usefulness of the isoleucine‐deprivation method for synchronization of androgen‐sensitive and androgen‐independent prostate cancer cells, and for examining the role of androgen and AR in progression of androgen‐sensitive prostate cancer cells from G₁ into S phase. © 2003 Wiley‐Liss, Inc.