Immunostaining of heat shock protein 27 (Hsp27) protein species on two‐dimensional electrophoresis (2‐DE) gels with enhanced sensitivity yields 59 spots reacting with anti‐Hsp27 antibodies. Recombinant Hsp27 exists in 2‐DE as two major protein species which comigrate in the human myocardial pattern with Hsp27 spots C754 and D899 as defined in the heart high‐performance 2‐DE database (http://www.mdc‐berlin.de/∼emu/heart/). Preparative electrophoresis of human myocardial proteins and analysis of the enriched mass range 20–30 kDa by 2‐DE revealed eight protein spots (C438, C582, C658, C697, C754, C595, C750) from the human myocardial database and a new spot not previously detected on silver‐stained gels. These spots were identified as Hsp27 protein species by enzymatic in‐gel‐digestion and analysis by matrix assisted laser desorption‐ionization (MALDI) peptide mass fingerprinting and, in part, MALDI‐post source decay sequencing of single fragments. Possible post‐translational modifications were investigated: immunostaining tests with anti‐phospho‐serine/‐threonine/‐tyrosine antibodies, although positive for other myocardial proteins, were negative for presumed Hsp27 protein species; likewise, periodate‐glycostaining assays and biotinylation screening did not detect modifications in the investigated Hsp27 protein species.