Rahn et al. (J. Appl. Physiol. 69: 1546-1548, 1990) showed that the gas pressure in a plethysmograph containing an intact egg oscillates in phase with electrocardiogram (ECG) and that this pressure variation could be used as a noninvasive way to determine the heart rate of an avian embryo. One possible mechanism to account for the pressure oscillation is the mechanical movement of the embryonic heart, which leads to volume shifts of gas within the plethysmograph. Another possibility is that the oscillation of gas pressure with heartbeat is pulsatile gas exchange resulting from pulsatile blood flow. If gas exchange were transiently stopped, a pressure signal dependent on gas exchange should disappear, while a pressure signal dependent on cardiovascular motion should persist. Using a number of late-age hen eggs (at days 15-20 of incubation), we tested these hypotheses by suddenly changing the gas composition surrounding an egg and measuring the effect of the pressure oscillation. We found that 1) after 5% CO2-95% N2 was flushed into the plethysmograph (presumably halting gas exchange), pressure oscillations went almost to zero and the ECG signal remained; after air was flushed back to the plethysmograph, the pressure signal returned to control level; 2) after 20% CO2-20% O2-60% N2 was flushed into the plethysmograph (presumably increasing net gas exchange), the pressure signal increased 2.5-fold compared with that in air; and 3) after 1% CO2-99% N2 was flushed into the plethysmograph (presumably reversing gas exchange), the oscillation pressure decreased to one-fourth of that in air and the phase of pressure relative to ECG reversed compared with the phase in air.(ABSTRACT TRUNCATED AT 250 WORDS)