A fluorescent derivative of ceramide, N-(6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminohexanoyl)-D-erythr o-sphingosine (C6-NBD-Cer), has been shown to label the Golgi apparatus of cultured cells (Lipsky, N. G., R. E. Pagano, Science 228, 745-747 (1985)). There is no unequivocal explanation for this Golgi labeling which is important in view of photolabeling of Golgi proteins which might be involved in metabolism, sorting and transport of (glyco)sphingolipids. To gain more insight into the mode of accumulation of this fluorescent ceramide analogue in the Golgi apparatus, we have synthesized two novel derivatives of C6-NBD-Cer, namely its 1-O-methyl and 3-O-methyl ether, and studied their uptake by and metabolism as well as intracellular distribution in cultured fibroblasts. Like C6-NBD-Cer both of these methyl ethers were able to diffuse across the plasma membrane at 7 degrees C and to label intracellular membranes. Within the first 30 min no conspicuous labeling of the Golgi apparatus was to be seen suggesting that all three ceramide analogues have no distinct affinity to this organelle. However, C6-NBD-Cer as well as the 3-O-methyl-C6-NBD-Cer slowly gave rise to labeling of Golgi membranes when the temperatures was maintained at 7 degrees C. With this increasing Golgi labeling a concomitant formation of C6-NBD-glucosylceramide and C6-NBD-sphingomyelin as well as 3-O-methyl-C6-NBD-sphingomyelin was observed. This demonstrates that the observed Golgi labeling is due to the formation of the respective fluorescent metabolites rather than to the fluorescent ceramide analogues themselves. This idea is consistent with our finding that when 1-O-methyl-C6-NBD-Cer was used neither formation of metabolites nor labeling of Golgi membranes could be observed even if the temperature was raised to 37 degrees C.