Muscle sympathetic nerve activity (MSA; peroneal nerve) and arterial and femoral venous plasma norepinephrine (NE) were studied in 10 volunteers at rest, during a relaxation procedure (RELAX), and during two mental challenges, a word identification test (WIT) and a color word test (CWT). [3H]NE infusions were used to assess NE spillover to and clearance from plasma. Net NE overflow from the leg was calculated. RELAX reduced MSA and femoral venous NE concentrations. CWT increased blood pressure, cardiac output (thermodilution), and calf flow and reduced systemic vascular resistance. Responses to WIT were less marked. CWT increased MSA by 25%, femoral venous NE concentrations by 25%, and NE overflow from the leg by 26% at 3 min. Fractional epinephrine and [3H]NE extractions were flow related and decreased during CWT. The arterial contribution to femoral venous NE (about half) increased by 10% during CWT. Arterial NE levels and spillover increased, but NE clearance was unchanged. Femoral venous NE concentrations and NE spillover (not based on flow measurements) and regional NE overflow correlated with MSA. Thus NE concentrations in plasma reflect spillover rather than clearance at rest and during mental challenge. Biochemical and neurophysiological indexes of sympathetic activity correlate when assessed in the same region. Mental stress increases sympathetic activity in leg muscle.