A hop-specific cDNA library from glandular tissue-enriched hop cones was screened for Myb transcription factors. cDNA encoding for R2R3 Myb, designated HlMyb3, was cloned and characterized. According to the amino acid (aa) sequence, HlMyb3 shows the highest homology to GhMyb5 from cotton and is unrelated to the previously characterized HlMyb1 from the hop. Southern blot analyses indicated that HlMyb3 is a unique gene, which was detected in various Humulus lupulus cultivars, but not in Humulus japonicus. Reverse transcription and real-time PCR revealed the highest levels of HlMyb3 mRNA in hop cones at a late stage of maturation and in colored petiole epidermis, while the lowest levels were observed in hop flowers. Two alternative open reading frames starting in the N-terminal domain of HlMyb3, encoding for proteins having 269 and 265 amino acids with apparent molecular masses of 30.3 and 29.9 kDa, respectively, were analyzed as transgenes that were overexpressed in Arabidopsis thaliana, Nicotiana benthamiana, and Petunia hybrida plants. Transformation with the longer 269 aa variant designated l-HlMyb3 led to a flowering delay and to a strong inhibition of seed germination in A. thaliana. Nearly complete flower sterility, dwarfing, and leaf curling of P. hybrida and N. benthamiana l-HlMyb3 transgenotes were noted. On the contrary, the shorter 265-aa-encoding s-HlMyb3 transgene led in A. thaliana to the stimulation of initial seed germination, to fast initiation of the lateral roots, and to quite specific branching phenotypes with many long lateral stems formed at angles near 90 degrees . Limited plant sterility but growth stimulation and rather branched phenotypes were evident for s-HlMyb3 transgenotes of P. hybrida and N. benthamiana. It was found that both HlMyb3 transgenes interfere in the accumulation and composition of flavonol glycosides and phenolic acids in transformed plants. These effects on heterologous transgenotes suggest that the HlMyb3 gene may influence hop morphogenesis, as well as metabolome composition during lupulin gland maturation.