The aim was to determine whether follicle growth in cattle is accompanied by changes in levels of inhibin-A (inh-A), activin-A (act-A) and different Mr isoforms of follistatin (FS) in bovine follicular fluid (bFF), reflecting differential roles of these proteins during folliculogenesis. Follicles (n=146) from 2–20 mm diameter were dissected from ovaries of ~40 cattle. Immunoassays were used to measure total FS, act-A, inh-A, oestradiol (E) and progesterone (P) levels; immunoblotting was used to quantify the relative abundance of different FS isoforms. Follicle growth from 2–6 mm was associated with a 6-fold increase in inh-A and 30-fold increase in act-A; FS remained uniformly high from 2–10 mm. From 6–2 mm, inh-A remained high while act-A and FS fell 3-fold and 2-fold, respectively. Act-A/FS ratio increased 20-fold from 2–6 mm before falling slightly through to 20 mm. Act-A/inh-A ratio increased 6-fold from 2–6 mm before falling 2-fold from 6 to 17–20 mm. These findings imply a marked increase in relative activin ‘tone’ around the stage at which dominant follicle selection occurs. When larger follicles (13–20 mm) were subdivided according to E/P ratio, those with high ( > 5) E/P ratio had lower (2-fold; P < 0.001) levels of inh-A and act-A in comparison to follicles with low ( < 5) E/P ratio, but there were no significant differences in FS, act-A/inh-A ratio or act-A/FS ratio. Thus follicle size, but not oestrogenic status, has a major influence on the intrafollicular balance between act-A and its opposing factors, inh-A and FS. Six FS isoforms were detected in bFF (apparent Mr: 65, 41, 37, 35, 33 and 31 kDa) averaging 6, 13, 24, 26, 13 and 17% respectively of total FS. During growth from 2–20 mm the proportion of total FS represented by 65, 41 and 37 kDa isoforms increased ~2-fold while the proportion represented by the 33 and 31 kDa isoforms decreased by 3-fold and 1.6-fold, respectively. Treatment of bovine granulosa cells in vitro with FSH and IGF alone or in combination increased total FS secretion up to 12-fold but did not affect the relative abundance of the five different FS isoforms detected. While the functional significance of the intriguing shift in FS isoform abundance in bFF during follicle development remains to be established, we have shown that a marked increase in intrafollicular activin ‘tone’ accompanies bovine follicle growth from 3–6 mm, corresponding to the stage at which the FSH-dependent follicle selection mechanism operates in this species.