To investigate the biochemical components of egg-hatch in the body louse, Pediculus humanus, egg-shell-washings (ESW) were collected during the first 2 h post-hatching and analysed by gelatin SDS-PAGE. These ESW contained proteases with molecular mass in the range of 25–100 kDa; the most abundant proteases were ~25 kDa. The 3 main regions of protease activity in the one-dimensional gelatin SDS-PAGE gels resolved to at least 23 distinct regions of protease activity when analysed by two-dimensional gelatin SDS-PAGE, with iso-electric points spread over the entire 3 to 10 pH range. Mechanistic characterization indicated that the ESW contained proteases of the metallo-class, inhibited by both 1,10-phenanthroline and EDTA. Several protease inhibitors were tested for their ability to inhibit louse egg-hatch in vitro. The metalloprotease inhibitor 1,10-phenanthroline and the aminopeptidase inhibitor bestatin significantly inhibited (P<0·05) louse egg-hatch (100% and 58%, respectively). The presence of metalloproteases at the time of egg-hatch and the inhibition of egg-hatch in P. humanus by metalloprotease inhibitors suggests a crucial role for these proteases in the hatching of this medically important parasite.