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Laboratory-created small-molecule-dependent inteins enable protein structure and function to be controlled posttranslationally in living cells. Previously we evolved inteins that splice efficiently in Saccharomyces cerevisiae only in the presence of the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). In mammalian cells, however, these inteins exhibited lower splicing efficiencies and slower splicing in the presence of 4-HT, as well as higher background splicing in the absence of 4-HT. Here we further evolved ligand-dependent inteins in yeast at 30°C and 37°C. The resulting second-generation evolved inteins exhibit substantially improved splicing yields and kinetics. The improvements carried over to mammalian cells, in which the newly evolved inteins spliced with substantially greater (∼2- to 8-fold) efficiency while maintaining low background splicing levels. These second-generation inteins augment the promise of ligand-dependent protein splicing for probing protein function in mammalian cells.