The Golgi complex, a membranous organelle with important functions in membrane traffic and macromolecular synthesis, has been stained in living cells with a fluorescent sphingolipid. Cells were first incubated with liposomes containing N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine (C6-NBD-ceramide), or with a bovine serum albumin complex of the fluorescent lipid, and then examined by fluorescence microscopy. An intensely fluorescent perinuclear structure was identified as the Golgi apparatus by its colocalization with known Golgi markers in fixed cells. C6-NBD-ceramide was used to observe the morphology of the Golgi apparatus in living cells in the presence or absence of monensin or Colcemid, and during mitosis. In all cases, C6-NBD-ceramide revealed a Golgi apparatus in the living cell that was identical to that obtained with conventional procedures that require fixation.