Stacking fault energies (SFE) were determined in additively manufactured (AM) stainless steel (SS 316 L) and equiatomic CrCoNi medium-entropy alloys. AM specimens were fabricated via directed energy deposition and tensile loaded at room temperature. In situ neutron diffraction was performed to obtain a number of faulting-embedded diffraction peaks simultaneously from a set of (hkl) grains during deformation. The peak profiles diffracted from imperfect crystal structures were analyzed to correlate stacking fault probabilities and mean-square lattice strains to the SFE. The result shows that averaged SFEs are 32.8 mJ/m 2 for the AM SS 316 L and 15.1 mJ/m 2 for the AM crconi alloys. Meanwhile, during deformation, the SFE varies from 46 to 21 mJ/m 2 (AM SS 316 L) and 24 to 11 mJ/m 2 (AM crconi) from initial to stabilized stages, respectively. The transient SFEs are attributed to the deformation activity changes from dislocation slip to twinning as straining. The twinning deformation substructure and atomic stacking faults were confirmed by electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM). The significant variance of the SFE suggests the critical twinning stress as 830 ± 25 MPa for the AM SS 316 L and 790 ± 40 MPa for AM CrCoNi, respectively.Excellent combination of strength, ductility, and toughness has been found in an equiatomic, face-centered-cubic CrCoNiFeMn high-entropy alloys (HEA) 1 . The reason of the exceptional properties at cryogenic temperature has been mainly attributed to the evolution of the nanoscale twinning under plastic deformation, so-called twinning-induced plasticity 2,3 . Compared to the HEA, superior mechanical properties of CrCoNi medium-entropy alloys (MEA) have been recently reported at both room and cryogenic temperature 4-13 . High attention has been focused on the evolution of the twinning substructure and/or a new phase with hexagonal close packed structure instead of the initial deformation mode of the dislocation slip in MEAs 6-9 . Systematic examinations of the substructure elucidate that the critical twinning stress of 790 ± 100 MPa reaches at the earlier strain of 9.7-12.9% for CrCoNi MEA than 720 ± 30 MPa at ~25% for CrCoNiFeMn HEA because of higher yield strength and work hardening rate with larger shear modulus of the MEA 8-10 . Earlier formation of the nano-twinning and its activation over a more extended strain range is of importance accepted as the reason of the exceptional strength-ductility-toughness combination in MEA.Stacking fault energy (SFE) has been accepted as a responsible parameter to determine the deformation schemes, which is typically by slip (>45 mJ/m 2 ) to twinning (20-45 mJ/m 2 ) and/or phase transformation (<20 mJ/m 2 ) as often reported in austenitic stainless steels [14][15][16][17][18][19][20][21] . The SFE is defined as the energy per fault area by www.nature.com/scientificreports www.nature.com/scientificreports/ SFE ranges 11-24 mJ/m 2 , b p is the magnitude of the Burgers vector of partials (0.146 nm), G is the shear...