Smoking is a serious public health problem, and accurate real-time assessment of risk factors associated with smoking is critical to understanding smoking relapse. Negative affect is often described as a critical risk factor related to smoking relapse, and ecological momentary assessment (EMA) methods have been widely used to study real-time relations between negative affect and smoking. However, the factor structure of momentary negative affect ratings is unknown. The current investigation examined the multilevel factor structure and internal consistency of an EMA measure of negative affect. Daily assessments were collected for 1 week prequit and 3 weeks postquit from 113 adult daily smokers receiving nicotine replacement therapy and counseling to quit smoking. Results supported a 2-factor model with correlated but distinct agitation and distress factors, rather than a single-factor model of negative affect. The agitation factor was indicated by these items: impatient, tense/anxious, restless. The distress factor was indicated by these items: sad/depressed, upset, distressed. The 2-factor model had acceptable model fit and consistent factor loadings across 3 separate cessation phases: prequit, postquit with recent smoking, and postquit without recent smoking. The 2 factors were highly correlated, showed good internal consistency, and showed strong associations with theoretically relevant smoking and affect variables. Agitation was more strongly related to urge to smoke, and distress was more strongly related to recent stress. This study provides support for a 2-factor model of an EMA measure of negative affect and highlights distinct facets that may be useful for future investigations of affect and smoking.