As part of a programme to determine whether root development requires a pathway involving KNOTTED-like homeobox ( KNOX ) genes, we cloned the KNAT6 cDNA, a member of the KNOX gene family in Arabidopsis, from root tissue. The KNAT6 cDNA is represented by two isoforms, suggesting that the KNAT6 transcript is subject to alternative splicing. Promoter-reporter and RT-PCR analysis confirms expression of the KNAT6 gene in roots, and in particular in the phloem tissue close to the site of lateral root initiation, though not in the primary or lateral root meristem. The KNAT6 gene promoter activity is altered in pattern by treatment with exogenous auxin and in the rooty mutant background, with expression shifting distally in the root. In contrast, exogenous cytokinin suppresses KNAT6 promoter activity. KNAT6::GFP fusion protein is localized to the nucleus, consistent with the predicted function of KNAT6 as a transcription factor. Over-expression of the more abundant shorter isoform of the KNAT6 cDNA, but not of the less abundant longer form, leads to a lobed leaf phenotype, but no consistently aberrant root phenotype. Down-regulation of KNAT6 expression by RNA interference was associated with an increased total number of lateral roots. These data indicate a role for KNAT6 in modulating lateral root formation.