Our aim was to analyze the impact of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on bladder cancer cell growth in vitro. RT-4, TCCSUP, UMUC-3, and RT-112 bladder cancer cells were treated with VPA (0.125-1 mmol/l) without and with preincubation periods of 3 and 5 days. Controls remained untreated. Tumor cell growth, cell cycle progression, and cell cycle-regulating proteins were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and western blotting, respectively. Effects of VPA on histone H3 and H4 acetylation and HDAC3 and HDAC4 were also determined. Without preincubation, no tumor cell growth reduction was observed with 0.125 and 0.25 mmol/l VPA in TCCSUP, UMUC-3, and RT-112 cells, whereas 0.5 and 1 mmol/l VPA diminished the cell number significantly. VPA (0.25 mmol/l) did exert tumor growth-blocking effects after a 3-day preincubation. To achieve antitumor effects with VPA (0.125 mmol/l), a 5-day preincubation was necessary. A 3-day or 5-day preincubation was also necessary to distinctly delay cell cycle progression, with maximum effects at VPA (1 mmol/l). After the 5-day preincubation, the cell cycle-regulating proteins cdk1, cdk2, cdk4, and cyclins B, D1, and E were reduced, whereas p27 was enhanced. Diminished HDAC3 and 4 expression induced by VPA was accompanied by elevated acetylation of H3 and H4. VPA exerted growth-blocking properties on a panel of bladder cancer cell lines, commensurate with dose and exposure time. Long-term application induced much stronger effects than did shorter application and should be considered when designing therapeutic strategies for treating bladder carcinoma.