The present study examines the genetic and environmental etiology of the associations among respiratory sinus arrhythmia (RSA), heart rate (HR), skin conductance level (SCL), and non-specific skin conductance responses (NS-SCR)—measures that purportedly index the parasympathetic and sympathetic branches of the autonomic nervous system. The sample was drawn from a cohort of 1,219 preadolescent twins (aged 9–10). Multivariate analyses of the data were conducted using structural equation modeling. Almost all genetic and environmental influences on the measures acted through two latent factors. The first latent factor was largely responsible for the variance in heart rate, SCL and NS-SCR, reflecting sympathetic activity, and its proportions of variance due to genetic and shared environmental influences were 27 and 28% in males, and 31 and 41% in females, respectively. The second latent factor accounted for the variance in RSA and heart rate, reflecting parasympathetic activity; genetic and shared environmental factors explained 27 and 23% of the variance in males, respectively, and 35 and 18% of the variance in females. Measurement-specific genetic effects accounted for 14–27% of the total variance in RSA and SCL, and measurement- specific shared environmental effects accounted for 10–12% in SCL. In general, the validity of separate sympathetic and parasympathetic constructs was supported.