By immunocytochemistry the distribution and developmental expression of the small EF-hand calcium-binding protein calsensin in the peripheral (PNS) and central nervous system (CNS) of the three hirudinid leech species Haemopis, Hirudo, and Macrobdella was compared. Labeling with calsensin-specific antibodies demonstrated that there was a pronounced difference in the distribution of calsensin immunoreactivity in the CNS of these leeches. In Haemopis more than 70 neurons were labeled, whereas the number in Hirudo was 51 and in Macrobdella only 8. Furthermore, the expression of calsensin in identified cells common to all three leech species also differed. Immunoblot analysis indicated that this variability was not likely to be due to multiple proteins or isoforms being recognized by the calsensin antibody. Labeling of embryos in various stages of development shows that the ontogeny of calsensin expression in the CNS is a gradual process with some neurons expressing calsensin immediately after completion of neurogenesis, about one-third of the way through embryogenesis, and others expressing calsensin only postembryonically. In contrast to the variability in the pattern and temporal expression by CNS neurons, the early embryonic calsensin expression in a small subgroup of sensillar PNS neurons was a shared feature by all three leech species. These findings suggest that calsensin may have different functional properties in CNS and PNS neurons.