OBJECTIVE: To determine whether metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction. DESIGN: Experimental study. SETTING: Academic research laboratories. PATIENT(S): None. INTERVENTION(S): Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n = 52) were compared with wild-type (WT) oocytes (n = 55) as a model of severe oocyte dysfunction. Oocytes from old mice (1 year old; n = 29) were compared with oocytes from young mice (12 weeks old; n = 35) as a model of mild oocyte dysfunction. MAIN OUTCOME MEASURE(S): FLIM was used to measure the naturally occurring nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ(1)) and long (τ(2)) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). Reactive oxygen species (ROS) levels and blastocyst development rates were measured to assess illumination safety. RESULT(S): In Clpp-knockout oocytes compared with WT, FAD τ(1) and τ(2) were longer and I was higher, NADH τ(2) was longer, and F was lower. In old oocytes compared with young ones, FAD τ(1) was longer and I was lower, NADH τ(1) and τ(2) were shorter, and I and F were lower. FLIM did not affect ROS levels or blastocyst development rates. CONCLUSION(S): FLIM parameters exhibit strong differentiation between Clpp-knockout versus WT, and old versus young oocytes. FLIM could potentially be used as a noninvasive tool to assess mitochondrial function in oocytes.