We describe the properties of npq4-9, a new mutant of Arabidopsis thaliana (L.) Heynh. with reduced nonphotochemical quenching (NPQ) capacity that possesses a single amino acid substitution in the PsbS gene encoding PSII-S, a ubiquitous pigment-binding protein associated with photosystem II (PSII) of higher plants. Growth, photosynthetic pigment contents, and levels of the major PSII antenna proteins were not affected by npq4-9. Although the extent of de-epoxidatin of violaxanthin to antheraxanthin plus zeaxanthin for leaves displaying the mutant phenotype equaled or exceeded that observed for the wild type, the relative effectiveness of de-epoxidized xanthophylls in promoting NPQ was consistently lower for the mutant. Energy partitioning in PSII was analyzed in terms of the competition for singlet chlorophyll a among the processes of fluorescence, thermal dissipation, and photochemistry. The key processes of NPQ and photochemistry in open PSII centers are represented by the relative in vivo rate constants kN and kP0, respectively. The magnitude of kP0 in normal leaves declined only slightly with increasing kN, consistent with localization of NPQ primarily in the antenna complex. Conversely, a highly significant linear decline in kP0 with increasing kN was observed for the mutant, consistent with a role for the PSII reaction center in the NPQ mechanism. Although the PSII absorption cross-section for white light was not significantly different relative to that of the wild type, PSII quantum yield was significantly lower in the mutant. The resulting lower capacity for linear electron transport in the mutant primarily affected reduction of terminal acceptors other than CO2. Parallel measurements of fluorescence and in vivo absorbance at 820 nm indicated a consistently higher steady-state level of reduction of PSII acceptors and accumulation of P700+ for the mutant. This suggests that inter-photosystem electron transport in the mutant is restricted either by a higher transthylakoid delta pH or by diminished accessibility to reduced plastoquinone.