The objectives of the present study were to compare the effects of two regimens of serial blood sampling on the concentrations of hormones and ions during the ovulatory cycle of the domestic hen, and to examine the effectiveness of an indwelling vascular access device for repeated collection of blood samples. Single Comb White Leghorn hens were bled every 2 h over a period of 24 to 26 h, either from one oviposition to the next oviposition (OVIP-OVIP), or from 10 h prior to ovulation until the same time 24 h later (AFTN-AFTN). Whole blood was analyzed for ionized calcium concentration. Plasma was analyzed for total calcium, inorganic phosphorus, 1,25-dihydroxycholecalciferol, estradiol-17beta, and progesterone concentrations. The OVIP-OVIP regimen, using oviposition as a reference point, provided more accurate measurements of ionized calcium, inorganic phosphorus, and estradiol-17beta than did the AFTN-AFTN regimen. Either bleeding regimen was suitable for observing the patterns of 1,25-dihydroxycholecalciferol and progesterone concentrations. The decrease in bound calcium concentration observed with both regimens appeared to be an artifact of repeated blood sampling. The chance of a bird laying an egg following her second oviposition was lower following the OVIP-OVIP regimen than the AFTN-AFTN regimen. The vascular access device was a helpful tool in procuring multiple blood samples for measurement of ions and hormones during the ovulatory cycle of the domestic fowl.