At least two transport systems serve for the entry of alanine, glycine, and serine into Escherichia coli. One of these systems serves mainly for glycine, d-alanine, and d-serine and to some extent for l-alanine, whereas the second serves for l-alanine and perhaps l-serine. These two transport systems have been characterized by kinetic studies and by inhibition analysis. Reciprocal plots for l-alanine entry are distinctly biphasic, giving rise to K(m) values of about 2 and 27 muM. The major route of glycine entry can be described by a single K(m) value of about 4 muM. A higher K(m) value for glycine of around 70 to 100 muM shows that other routes of entry may serve at high concentrations of amino acid. The glycine, d-serine and d-alanine transport system is defective in a d-serine-resistant mutant, strain EM1302. The mutation, dagA, is recessive in dagA/dagA(+) merodiploids and is 7 to 12% linked by phage P1 transduction to the pyrB locus of E. coli. E. coli with the dagA mutation are unable to utilize d-alanine as a carbon source, providing an additional basis for selecting such mutants. The remaining l-alanine uptake in dagA mutants is subject to inhibition by l-serine, l-threonine, and l-leucine. It is also sensitive to osmotic shock treatment and repressed by growth of the cells on l-leucine. It appears from a comparison of the properties of the second l-alanine system with those of the leucine, isoleucine, and valine system (LIV system) that the LIV system also serves for the transport of l-alanine and l-threonine and perhaps l-serine.