Molecular, genetic, and electrophysiological evidence indicates that at least one of the plant Glu receptor-like molecules, GLR3.4, functions as an amino acid–gated Ca2+ channel at the plasma membrane. The aspect of plant physiology, growth, or development to which GLR3.4 contributes is an open question. Protein localization studies performed here provide important information. In roots, GLR3.4 and the related GLR3.2 protein were present primarily in the phloem, especially in the vicinity of the sieve plates. GLR3.3 was expressed in most cells of the growing primary root but was not enriched in the phloem, including the sieve plate area. GLR3.2 and GLR3.4 physically interacted with each other better than with themselves as evidenced by a biophotonic assay performed in human embryonic kidney cells and Nicotiana benthamiana leaf cells. GLR3.3 interacted poorly with itself or the other two GLRs. Mutations in GLR3.2, GLR3.4, or GLR3.2 and GLR3.4 caused the same and equally severe phenotype, namely, a large overproduction and aberrant placement of lateral root primordia. Loss of GLR3.3 did not affect lateral root primordia. These results support the hypothesis that apoplastic amino acids acting through heteromeric GLR3.2/GLR3.4 channels affect lateral root development via Ca2+ signaling in the phloem.