The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops.