The cooling effect in molecular beam (MB) sampling from low-pressure flames and gas mixtures was investigated. Although the MB method is often used to study the flame structure of low-pressure flames, typically combined with mass spectrometric (MS) detection, it is poorly characterized. The temperature of the MB must be known, especially if species concentrations are to be measured with spectroscopic methods, like resonance-enhanced multiphoton ionization (REMPI) spectroscopy. In the present study, two independent MBMS instruments, which are very similar to those used previously by different groups, were investigated starting with pressures of 40 and 50 mbar in the burner chamber. The rotational temperatures of NO and benzene were determined using REMPI spectroscopy for different initial conditions; the ions were separated by time-of-flight mass spectrometers. Two REMPI excitation schemes were applied: for NO the first step was always the excitation of the A-X transition near 225 nm, while benzene was excited and ionized at wavelengths near 259 nm. Unexpectedly, molecular beams from cold-gas flows were cooled very slightly by 10%-25%. In the molecular beams derived from low-pressure flames, the cooling effect was stronger, with final rotational temperatures of 300-400 K, but the MB temperature was virtually independent of the initial temperature. A possible explanation of this finding would be that the cooling takes place to a large extent by wall collisions within the nozzle and to a lesser degree by intermolecular collisions.