Color Doppler ultrasound measurements of luteal blood flow (LBF) may serve as an accurate way to estimate corpus luteum (CL) function. Luteal volume (r = 0.69) and luteal blood flow (r = 0.71) are highly correlated with circulating concentrations of progesterone. Our aim was to determine the effect of different doses of PGF_2α on blood flow measurements of immature and mature CL. This study utilized n = 37 11- to 12-mo-old Holstein heifers. Heifers were synchronized with 500 µg of cloprostenol on d –8; 100 µg of GnRH on d –6; and 100 µg of GnRH on d 0. Only heifers that responded to each injection were utilized. On d 4 (day of treatment) all heifers had ovaries with both a d 4 and 10 CL. On day of treatment, heifers were randomly assigned to 5 treatments: full dose of cloprostenol (500 µg; n = 8), 1/2 dose (250 µg; n = 8), 1/4 dose (125 µg; n = 8), positive control (4 treatments of 500 µg cloprostenol 24 h apart; n = 5), or a negative control (no treatment; n = 8). Color Doppler was used to assess LBF (numbers of color pixels) before treatments and at 1, 48, 96, 144, 192 h post-treatment. Negative controls had less LBF 1 h following treatment compared with all other treatments for the d 10 CL. Negative controls had greater LBF 48, 96, 144, and 192 h following treatment compared with all other treatments in d 10 CL. Positive controls and heifers that received a full dose had less LBF compared with 1/2 and 1/4 dose groups at 48, 96, 144, and 192 h following treatment in d 10 CL. There was no difference at 48, 96, 144, and 192 h following treatment in 1/4 and 1/2 dose groups in the d 10 CL. There were no differences in LBF in the d 4 CL at 1 or 48 h but the positive control had less LBF at 96 and 144 h compared with all other treatments. There was no difference in the positive control compared with the full dose at 192 h in the d 4 CL. The full dose had less LBF at 192 h compared with the 1/2, 1/4, and negative control in the d 4 CL. The differential effects of treatments indicated that color Doppler might be an accurate assessment of luteal function before and following PGF_2α.