The isolation of high-quality RNA is a prerequisite for gene expression studies. RNA quality is of special relevance if PCR-based strategies such as cDNA-AFLP or RT-PCR are followed. Our molecular investigations of hop (Humulus lupulus L.) focus on genes that determine the biosynthesis of prenylflavonoids with interesting biological activities which accumulate in the lupulin glands. However, optimized protocols for RNA extraction from hop cones are not available. In this study, the RNeasy midi kit protocol was modified to isolate high amounts of total RNA from fresh and freeze-dried hop tissues, specifically leaves, female inflorescences, and lupulin-enriched hop cone fractions. The main difficulties in obtaining high RNA yields were related to specific features of hop, including the abundance of secondary metabolites and their accumulation in the sticky lupulin glands. The introduction of a number of modifications into the RNeasy midi kit protocol resulted in RNA of high quality, as assessed by spectrophotometry and electrophoresis in agarose gels. The protocol developed is currently being used to produce cDNA-AFLP fingerprints of hop cones and leaves for genetic screening. Furthermore, the method could provide RNA for further molecular studies, including Northern blot hybridizations and reverse transcription-polymerase chain reactions.