In Arabidopsis, the small signaling peptide (peptide hormone) RALF34 is involved in the gene regulatory network of lateral root initiation. In this study, we aimed to understand the nature of the signals induced by RALF34 in the non-model plant cucumber (Cucumis sativus), where lateral root primordia are induced in the apical meristem of the parental root. The RALF family members of cucumber were identified using phylogenetic analysis. The sequence of events involved in the initiation and development of lateral root primordia in cucumber was examined in detail. To elucidate the role of the small signaling peptideCsRALF34 and its receptorCsTHESEUS1 in the initial stages of lateral root formation in the parental root meristem in cucumber, we studied the expression patterns of both genes, as well as the localization and transport of theCsRALF34 peptide.CsRALF34is expressed in all plant organs.CsRALF34seems to differ fromAtRALF34in that its expression is not regulated by auxin. The expression ofAtRALF34,as well asCsRALF34,is regulated in part by ethylene.CsTHESEUS1is expressed constitutively in cucumber root tissues. Our data suggest thatCsRALF34 acts in a non-cell-autonomous manner and is not involved in lateral root initiation in cucumber.