The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin cysteine 374 by tetramethyl-rhodamine was reported to make actin non-polymerizable and allowed to solve the crystal structures of ADP-actin and AMPPNP-actin. TMR-actin has also been used to solve the structure of actin in complex with the FH2 domain of formin mDia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerizes in very short filaments that are destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP are only one order of magnitude higher than for unlabeled actin. The functional interactions of actin with capping proteins, formin, ADF/cofilin and the VCA-Arp2/3 filament branching machinery are profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of the actin molecule in the ATP-bound or ADP-bound state which allow its specific adaptative recognition by regulatory proteins and which determine its function.