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This report covers the two-photon activation and excitation properties of the PA-GFP, a photoactivatable variant of the Aequorea victoria green fluorescent protein in the spectral region from 720 to 920nm. It is known from this special form of the molecule that it has an increased level of fluorescence emission when excited at 488nm after irradiation at λ ∼ 413nm, under single-photon excitation conditions. Here, we show that upon two-photon irradiation, PA-GFP yields activation in the spectral region from 720 to 840nm. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein. Additionally, when comparing the conventional photoactivation at λ=405nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.