The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone–bovine serum albumin–fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 ± 4%), but this labelling was markedly reduced (33 ± 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 ± 10% (onset) to 59 ± 7% by 5 h, where it plateaued. Progesterone (P₄) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 µg/mL P₄ corresponding to 10 ± 5%, 16 ± 9%, 23 ± 7% and 30 ± 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P₄-induced AR (12 ± 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P₄-induced AR (10 µg/mL) in capacitated ejaculated spermatozoa from 19 ± 6% to 11 ± 4% (h151, 1 : 10) and 12 ± 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P₄ induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.