During a follicular wave in mares, the two largest follicles (F1 and F2) begin to deviate in diameter when F1 is a mean of 22.5 mm. The intrafollicular effects of pregnancy-associated plasma protein-A (PAPP-A), IGF-I, activin-A and vascular endothelial growth factor (VEGF) on other follicular-fluid factors during deviation were studied. In four treated groups (n = 7/group), a single dose of one of the four factors was injected into F2 when F1 was > or = 20.0 mm (expected beginning of deviation). In a control group (n = 7), F2 was injected with vehicle. One day after treatment, a sample of follicular fluid was taken from F1 and F2 of the control group and from F2 of the treated groups and was assayed for free IGF-I, oestradiol, androstenedione, activin-A, inhibin-A, follistatin and VEGF. In the control group, the means for all end points were significantly greater in F1 than in F2, except that concentrations of androstenedione were lower in F1 than in F2. The treatment effects for F2 were significant as follows: PAPP-A increased the concentrations of free IGF-I, inhibin-A, follistatin and VEGF and decreased the concentrations of androstenedione; IGF-I increased the concentration of inhibin-A and decreased the concentration of androstenedione; activin-A decreased the concentrations of follistatin and androstenedione and increased the diameter of F2; and VEGF increased the concentration of IGF-I and decreased the concentration of androstenedione. These results support the hypotheses that during deviation in mares PAPP-A increases the follicular-fluid concentrations of free IGF-I, follistatin responds to changes in follicular-fluid concentrations of activin-A, and VEGF affects the concentrations of other follicular-fluid factors.