The gain spectrum of a pulsed submillimeter (SMM) laser amplifier has been determined for the first time using single‐mode pump and submillimeter laser oscillators. The gain of a 385‐&mgr;m D2O laser amplifier was measured as a function of CO2laser pump power, D2O laser oscillator power, polarization, and D2O gas pressure. The use of single‐mode lasers permits a direct comparison with theory. Comparison with a theory which takes into account two‐photon contributions to the gain yields good qualitative agreement, in that theory accurately predicts the shape of the gain spectrum, including ac Stark effects and the dependence of the gain on polarization, but not the absolute magnitude of the gain.