Helium isotopes have been measured in fluids trapped in minerals from three mid-ocean ridge hydrothermal systems at bare-rock sites and at the sediment-covered site at Middle Valley, northern Juan de Fuca Ridge. Fluid-inclusion 3He/4He ratios from the bare-rock sites are indistinguishable from those of the vent fluids and are close to the local basalt values. Values for δ34S of the host sulfide are consistent with variable contributions of seawater sulfate S to the basalt-derived S in the hydrothermal fluids. This does not affect inclusion-hosted 3He/4He because of the high He concentration of the hydrothermal fluids relative to seawater. Fluid-inclusion 3He/4He ratios from Middle Valley are a mixture of basalt-derived He and radiogenic He acquired from pore fluids in the overlying Pleistocene turbidite sediments after seawater-basalt interaction. The radiogenic He accompanies isotopicalty heavy S, demonstrating that He isotopes may be used to distinguish pore fluids from seawater in hydrothermal systems.