Escherichia coli cells in peptone water were pressurized at 300 MPa at ambient temperature with no holding time (pulse series) and with a total holding duration of 300 s for single- (300 s x 1 pulse) and multi-pulsed (150 s x 2 pulses, 100 s x 3 pulses, 75 s x 4 pulses, 60 s x 5 pulses, 50 s x 6 pulses and 30 s x 10 pulses) high hydrostatic pressure (HHP) treatments. Multi-pulsed HHP treatment with no holding time indicated that as the pulse number increased the number of inactivated and injured cells also increased. Holding time had significant effect on the inactivation of E. coli. There was low inactivation difference between single- and multi-pulsed HHP treatments with holding time. Escherichia coli cells showed at least 1.6 log(10) more reduction on selective medium than the non-selective medium indicating that more than 95 % of the survivors severely injured for both single- and multi-pulsed treatments with holding time. Although the inactivation difference was low between single- and multi-pulsed HHP treatments, storage at 4 A degrees C revealed that there was less recovery from injury for multi-pulsed HHP treatment.