Abstract: This study investigated the physiological and biochemical changes during postharvest senescence of two broccoli (Brassica oleracea L.) cultivars (‘Hartland’ and ‘Sairin’). A parallel trend in weight loss, color change and respiration rate was observed in both cultivars. However, ‘Sairin’ exhibited higher total weight loss and rate of respiration and lower hue angle value. Glutamine synthetase (GS, EC 6.3.1.2) activity decreased in the florets of both cultivars while an increasing glutamate dehydrogenase (GDH, EC 1.4.1.2) activity was noted in the florets of ‘Hartland’. On the other hand, GDH activity was not detected in the floret of ‘Sairin’. In the branchlet portion of both cultivars, GS activity was nearly unchanged except for a transient increase during the first 24 h of storage. Initial GDH-aminating and deaminating activities were significantly higher in this portion but were not maintained until the end of the storage period. Ammonia accumulated largely in the floret portion which accounted for about 87 and 93% of initial contents in ‘Hartland’ and ‘Sairin’, respectively. On the contrary, ammonia concentration in the branchlet portion of both cultivars remained almost unchanged. Total amino acid in all portions of both cultivars increased as storage progressed. A highly significant negative correlation was observed between GS activity and ammonia and total amino acid accumulations while a highly significant positive correlation was evident between GDH activity and ammonia and total amino acid accumulations in the florets of ‘Hartland’. In the branchlet portions, only GDH-deamination showed significant negative and highly significant negative correlations with ammonia and total amino acid accumulations, respectively. Comparing the two cultivars, ‘Hartland’ has longer shelf life than ‘Sairin’. The shorter shelf life of ‘Sairin’ could be attributed to higher respiration rate and the deficiency of GDH activity.