We previously generated and characterized a genuine estrogen receptor (ER) β-null mouse line (named ERβ(ST)(L-/L-)) and showed that ERβ(ST)(L-/L-) mice were sterile, due to an ovulation impairment in females and to an unknown reason in males, as their reproductive organs and spermatozoid motility appeared normal. We report here an assessment of the sexual behavior of ERβ(ST)(L-/L-) null mice. We found that ERβ(ST)(L-/L-) males display mildly impaired sexual behavior and that ERβ(ST)(L-/L-) females are significantly less receptive and less attractive than wild-type (WT) females. Decreased attractivity is also exhibited by ERβAF2(0) but not by ERβAF1(0) mutant females (females devoid of either AF2 or AF1 activation function of ERβ). Interestingly, by using an odor preference test, we have determined that the low attractiveness of ERβ(ST)(L-/L-) and ERβAF2(0) females is related to a deficiency of a volatile chemosignal.