ABSTRACT. We studied the role of the autonomic nervous system in the regulation of heart rate variation (HRV) in 12 chronically instrumented neonatal lambs. HRV was quantified from ECG tracings by computing periodic HRV distributions at frequencies of 0.02-1.00 Hz, using power spectral analysis of heart rate, and also by HRV indices. Heart rate declined more during the 1st than the 2nd mo after birth. Multiple regression analysis showed that the heart rate responses to vagal and to @-adrenergic blockade had an independent negative association both with age and with the initial mean heart rate, whereas the overall HRV response had a positive association with age. Vagal blockade led to a 70-80% decrease in the beat-to-beat HRV in all lambs ( p < 0.001). The overall HRV indices decreased by 40-65% in lambs (< 30 d old ( p < 0.001) and about 30% in those >30 d old ( p < 0.05). In the power spectrum the greatest decrease was seen in the high-frequency components of HRV. @-Blockade led to a decrease of about 50% in all HRV ( p < 0.001) in the younger lambs, without frequency selection. In the older lambs, it had no effect on the beat-to-beat HRV, but the overall HRV (coefficient of variance) decreased maximally by 40% ( p < 0.01), with a significant reduction in the low-frequency components of HRV. These results suggest that in the regulation of HRV after birth dual control via the autonomic nervous system is most important. In the older lambs, developmental changes result in precise regulation of the fast heart rate fluctuations mainly by the vagal division, whereas the slow fluctuations are partially regulated by the vagal and @-adrenergic divisions. (Pediatv Res 27: 383-391, 1990) Abbreviations HRV, heart rate variation RMSM, root mean square of differences from the mean interval, overall HRV CV, coefficient of variation for RMSM, relative overall HRV RMSR, residual HRV (RMSM) after trend correction CVR, coefficient of variation for RMSR, relative residual HRV RMSSD, root mean square of successive interval differences, beat-to-beat HRV CVS, coefficient of variation for RMSSD, relative beat-tobeat HRV