The right hemisphere is assumed to play a unique role for pain sensitivity and negative affect. Pressure pain thresholds were assessed daily in eight right-handed participants over a 6-week period in order to obtain reliable measures of pain sensitivity unaffected by situational influences. In an additional session, cerebral laterality was assessed with behavioral and an EEG measures. Psychometric measures were used to examine emotionality (depression, neuroticism). Pain thresholds were lower on the left compared with the right hand, and pain thresholds increased within the first 3-measurement weeks. Enhanced pain sensitivity as reflected in weekly pain threshold was associated with a increased right frontal compared with left frontal brain activity as indicated by EEG, with a left-visual field advantage in the perception of emotional faces, and with increased negative affect (depression, neuroticism). In addition, a significant positive correlation between a relatively increased right frontal brain activity and depression was found. Correlations between pain thresholds and 'non-emotional' laterality measures (central or parietal EEG asymmetry, dichotic consonant-vocal-recall test) were not significant. We conclude that a right frontal brain hyperactivity might be a biological marker for enhanced pain sensitivity and negative affect.