5′‐Nucleotidase, purified to homogeneity from chicken gizzard using published procedures [Dieckhoff, J., Knebel, H., Heidemann, M. and Mannherz, H. G. (1985) Eur. J. Biochem. 151, 377–383] was incorporated into artificial phospholipid vesicles after prolonged dialysis against detergent‐free buffer or by a gel filtration procedure. After dialysis the obtained liposomes exhibit a mean diameter of 80 nm and contain 5′‐nucleotidase at random orientation, demonstrated by finding up to 50% of the total liposome‐incorporated AMPase activity to be cryptic, i.e. could only be measured after their permeabilization by addition of detergent. By affinity chromatography a phospholipid vesicle fraction could be obtained containing almost exclusively cryptic AMPase activity, thus representing the inside‐out orientation of 5′‐nucleotidase. Comparative analysis of physiochemical and enzymatic properties of 5′‐nucleotidase reveals differences between the detergent‐solubilized and the liposome‐incorporated 5′‐nucleotidase including a changed accessibility of the enzyme to polyclonal and monoclonal antibodies. Binding and AMPase inhibition studies with different polyclonal antibodies strongly indicate to the existence of a cytoplasmic domain of chicken gizzard 5′‐nucleotidase. F‐actin appears preferentially to interact with the cytoplasmic domain of liposome‐incorporated 5′‐nucleotidase.