We evaluated the presence and number of eosinophils at varying stages in the human corpus luteum from 27 ovaries of women at reproductive age. Eosinophils preferentially accumulated in dilated microvessels of the thecal layer transforming into septa of the corpus luteum. The granulosa layer under luteinization, the thecal layer, and haemorrhages in the former antrum each contained low, moderate and high numbers of extravasated eosinophils respectively. Eosinophils decreased rapidly during the stages of secretion and regression. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) systems were used to investigate the expression and regulation of the eosinophil-attracting chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin in granulosa cells obtained from follicular aspirates from women undergoing IVF. Contaminating leukocytes were determined by CD18 mRNA quantification. Granulosa cells expressed RANTES (n = 3; 43 +/- 14 pg/ml, mean +/- SEM). 4ss-phorbol-12-myristate-13-acetate (PMA; 211 +/- 53) and tumour necrosis factor alpha (TNFalpha) (238 +/- 59), but not interleukin (IL)-1 up-regulated RANTES at significant levels. In general, higher basal and stimulated RANTES mRNA and protein were found in cultures with higher CD18 mRNA levels than in those with lower levels. We found only traces of eotaxin mRNA and no eotaxin secretion, even in stimulated granulosa cell cultures, independently of leukocyte levels. Taken together, this is the first study demonstrating the selective presence of eosinophils in human periovulatory structures. RANTES, but not eotaxin, may play an active process in the accumulation of these cells.