We compared the accuracy and reproducibility of a hand-held portable ultrasound pachymeter, the Pach-Pen (Bio-Rad, Ophthalmic Division, Santa Ana, California); another ultrasound pachymeter, the DGH 1000 (DGH Technology, Inc., Frazer, Pennsylvania); and the Pro-Cem 4 endothelial specular microscope (Alcon-Surgical, Inc., Irvine, California). Each eye of 18 healthy human subjects was examined to determine corneal thickness using the three different instruments. For each instrument, five repeated measurements were obtained at each of five corneal locations (one central, four peripheral), for a total of 25 measurements per eye. The accuracy of the two ultrasound pachymeters was tested by comparing measurements obtained on specially designed test blocks of known thickness. The Pach-Pen was the more accurate of the two ultrasound pachymeters, with measurements within the range of 0.003 to 0.065 mm from the true thickness. The three instruments were most consistent in mean thickness in the center of the cornea. All three instruments showed excellent intraobserver reproducibility, as measured by reliability coefficients over 90%. Overall, the Pach-Pen pachymeter had high reproducibility, and produced more accurate measurements than the DGH 1000 pachymeter.