The in vitro interaction between the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytoskeletal elements is well documented. To verify this association within cells, the intracellular distribution of GAPDH under various metabolic conditions has been investigated in immunostained cells or cells expressing GAPDH as a GFP fusion protein. GAPDH was homogeneously distributed in the cytoplasm and no interaction of GAPDH with cytoskeletal elements, neither with microfilaments nor microtubules or intermediate filaments, was detectable. In living cells expressing GFP-GAPDH, stress fibres were excluded from the fluorescence. In contrast to proliferating cells, the cytoplasmic GAPDH of serum-depleted cells was not homogeneously distributed, but colocalised with stress fibres. The mechanism for stimulating this actin-binding affinity was independent of the NO-signalling pathway. The results support the idea of a specialised function for the interaction of GAPDH and cytoskeletal elements, rather than a general function, as e.g. microcompartmentalization of glycolytic enzymes.