A yeast mutant defective in permeases S1 and S2 which transport L-leucine was isolated from a parental strain already deficient in the general amino acid permease, GAP1. The mutant was selected as a spontaneous, trifluoroleucine-resistant (TFLR) strain. Full resistance depended upon the presence of two unlinked mutant genes designated let1 and let2. The let1 mutation completely inactivates the high-affinity leucine transport system defined kinetically as S1. Although the let2 mutation caused a marked decrease in the Jmax of the low-affinity transport system, S2, residual leucine transport in the let1 let2 gap1 mutant had the same KT as in the LET1 LET2 gap1 parent. The mutant exhibited a marked decrease in growth on minimal medium containing leucine, isoleucine or valine as a sole nitrogen source. Moreover, assimilation of methionine, phenylalanine, serine and threonine was decreased, whereas basic and acidic amino acids supported normal growth. This indicates that at least one of the leucine permeases has a fairly broad, but still limited, specificity. Reversion of the gap1 gene restored leucine transport. The revertant was sensitive to TFL when grown on proline but resistant when NH4+ was the nitrogen source. The previously published mutations (shr3, aat1, lup1 or raa) would not be related to either LET1 or LET2.