Circadian clock genes have the function of producing circadian rhythm. They are also implicated in the origin or development of many diseases such as cancers and neuropsychiatric diseases. The purpose of this study is to determine whether the 54-nucleotide repeat polymorphism of hPer3, one of the circadian clock genes, associates with heroin dependence. DNA samples were obtained from 209 Chinese heroin-dependent subjects and 249 Chinese healthy controls. The 54-nucleotide repeat polymorphism was detected by polymerase chain reaction and DNA agarose gel electrophoresis. The frequency of four-repeat allele was significantly higher (chi(2)= 10.64, P = 0.001; corrected for multiple tests, P = 0.003) in the mixed gender heroin-dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the mixed gender control group (four repeat: 0.81, five repeat: 0.19); the frequency of four-repeat allele was also significantly higher (chi(2)= 10.00, P = 0.002; corrected for multiple tests, P = 0.006) in the male heroin-dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the male control group (four repeat: 0.81, five repeat: 0.19); for females, no significant trend was observed with the 54-nucleotide repeat polymorphism between the heroin-dependent subject group and the control group. Our results suggest that the 54-nucleotide repeat polymorphism of hPer3 significantly associates with heroin dependence at the allele frequency level and may be a potential risk factor for the development of heroin dependence.