A device and method for calibration of pulse oximeters is disclosed. A standard specimen that has optical characteristics of hemoglobin of a known oxygen saturation is placed in the light path of a pulse oximeter probe. The image of a pulse is created by a filter that periodically increases and decreases the amount of light allowed to reach the sensor of the probe. In one embodiment, the filter comprises a pair of polarizing filters rotated with respect to one another under the control of a digital computer with a motor interface device. In another embodiment, a wedge of a light-attenuating medium, such as a cuvette containing oxygenated blood, is reciprocated in the light path, the wide portions of the wedge attenuating a greater amount of light than the narrow portions. Such a cuvette can include filler openings, with outwardly extending tubes at both the wide and narrow ends. For comparison testing, the cuvette can include a sample of blood from a test subject, the blood having been oxygenated to a known oxygen content. A light-attenuating layer, with light transmissivity characteristics that are additive to those of the filter, can be provided in the light path to simulate an abnormal blood condition, such as the presence of carboxyhemoglobin, methemoglobin, or venous blood.