Rat urine contains many volatile constituents that may be used for chemical communication. The levels of certain urinary volatiles are strongly dependent on the sex and endocrine status (e.g., puberty). We performed chemical and behavioral studies to identify the volatiles in adult male rat urine that attract mature females. Our results demonstrated that adult male rats have higher levels of 2-heptanone (2-HP), 4-methylphenol (4-MP), and 4-ethylphenol (4-EP) than prepubescent male rats; furthermore, female rats are more attracted to the odor of adult male rat urine than that of prepubescent males. When prepubescent rat urine was supplemented with 2-HP, 4-MP, and 4-EP to the levels found in adult male urine, the attractiveness of the urine to females was markedly enhanced. Our results suggested that this attraction is due to an increased level of chemosignaling.