The aim of the study was to assess the physiological reference values for systemic and intrafollicular placental growth factor (PlGF) concentrations in different categories of follicular sizes in cycling mares, according to progesterone (P4) and oestradiol (E2) patterns. Sixty ovaries were taken after slaughter from 30 clinically healthy mares. Regarding their size, the follicles were classified into three different categories, i.e. small (20–30 mm), medium-sized (31–40 mm) and large (≥41 mm), and follicular fluid (FF) was sampled from each single follicle. Intrafollicular PlGF concentrations were significantly increased in larger and medium-sized follicles compared to small follicles, and their values were 1.48 and 1.36 times higher than the systemic values, respectively. On the other hand, systemic PlGF concentrations were 1.3 times higher than those in the FF of follicles of small size. Intrafollicular P4 concentrations were significantly higher in larger follicles than in small ones, and their concentrations were 6.74 and 3.42 times higher than the systemic values, respectively. Intrafollicular E2 concentrations were significantly higher in large and medium-sized follicles than in follicles of small size, and their concentrations were 21.1, 15.4 and 8.35 times higher than the systemic values, respectively. Intrafollicular and systemic PlGF concentrations were strongly and positively correlated; nevertheless, no correlations between intrafollicular and systemic steroid hormones, PlGF and follicle diameters, PlGF and E2, or PlGF and P4 were observed. This represents the first study to characterise systemic and intrafollicular PlGF concentrations in cycling normal mares, providing evidence that the bioavailability of this factor in follicles of medium and large sizes was higher than in small follicles, independently of steroid hormone concentrations. Further studies are needed to assess the presumable implications of PlGF in follicular angiogenesis in mares, similar to those already observed in women and primates.