Two methods of temperature control of a dual-beam optical-tweezers system are compared. In the first method, we used a 975 nm infrared laser to raise the temperature 5.6°C/100mW in a nonheating (830 nm) optical trap. The temperature increment logarithmically decreases toward the periphery of the heating beam, causing a fluid convection of 8 μm/s inside a 180 μm thick microchamber. In the second method, heating or cooling fluid was pumped through copper jackets that were placed on the water immersion objectives on both sides of the microchamber to control its temperature from 4.5°C to 68°C. The temperature controlled by the second method was both stable and homogeneous, inducing little fluid convection that would disturb single-molecule applications. An analysis of the power spectrum of the thermal force on a trapped bead showed no detectable vibration due to the liquid circulation. In both methods, force was measured directly by sensors of the momentum flux of light, independent of environmental disturbances including refractive index changes that vary with temperature. The utility of the second method was demonstrated in single-molecule experiments by measuring the mechanical stretch of a 41 kbp λ double-stranded DNA at temperatures ranging from 8.4°C to 45.6°C. © 2005 by the Biophysical Society.