The electronic excited-state behavior of photosystem II (PSII) in Mantoniella squamata, as influenced by the xanthophyll cycle and the transthylakoid pH gradient (ΔpH), was examined in vivo. Mantoniella is distinguished from other photosynthetic organisms by two main features namely (1) a unique light-harvesting complex that serves both photosystems I (PSI) and II (PSII); and (2) a violaxanthin (V) cycle that undergoes only one de-epoxidation step in excess light to accumulate the monoepoxide antheraxanthin (A) as opposed to the epoxide-free zeaxanthin (Z). The cells were treated first with high light to induce the ΔpH and A accumulation, followed by herbicide-induced closure of PSII traps and a chilling treatment, to sustain and stabilize the ΔpH and nigericin-sensitive fluorescence level in the dark. De-epoxidation was controlled with subsaturating concentrations of dithiothreitol (DTT) and was 5–10 times more sensitive to DTT than higher plant thylakoids. The PSII energy dissipation involved two steps: (1) the pH activation of the xanthophyll binding site that was associated with a narrowing and slight attenuation of the main 2 ns (ns = 10⁻⁹ s) fluorescence lifetime distribution; and (2) the concentration-dependent binding of A to the activated binding site yielding a second distribution centered around 0.9 ns. Consistent with the model of Gilmore et al. (1998) (Biochemistry 37, 13 582–13 593), the fractional intensity of the 0.9 ns component depended almost entirely on the A concentration and correlated linearly with the decrease of the steady-state chlorophyll a fluorescence intensity.