The neuropeptide profiles of the two major neuro-endocrinological organs, brain and retrocerebral complex corpus cardiacum-corpus allatum (CC/CA) of adult beetles, Zophobas atratus Fabricius (Coleoptera:Tenebrionidae) were analyzed by a combination of high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time of flight tandem mass spectrometry (MALDI TOF/TOF MS). The homological semi-isolated heart bioassay was used to screen HPLC fractions for myotropic activity in tissues, revealing several cardiostimulatory and cardioinhibitory factors from both the brain and CC/CA. Analysis of HPLC fractions by MALDI-TOF MS identified seven mass ions that could be assigned to other known peptides: leucomyosuppressin (LMS), Tribolium castaneum pyrokinin 2, sulfakinin 1, myoinhibitory peptide 4, a truncated NVP-like peptide, Tenebrio molitor AKH and crustacean cardioactive peptide. In addition, two novel peptides, myosuppressin (pEDVEHVFLRFa), which differs from LMS by one amino acid (E for D at position 4) and pyrokinin-like peptide (LPHYTPRLa) were also identified. To establish cardioactive properties of some of the identified peptides, chemical synthesis was carried out and their activities were tested using the heart bioassay.