Accurate estimation of krill biomass by acoustic techniques is dependant upon a number of factors including, fundamentally, accurate echo-sounder calibration. The Simrad EK500 scientific echo-sounder used aboard RRS James Clark Ross is regularly calibrated at South Georgia before and after krill surveys, and exhibits acceptable temporal stability in system gain settings. Between Antarctic seasons, this echo-sounder has additionally been calibrated in temperate European waters and, under these conditions, calibrated gain settings differ markedly from those applied in the Antarctic, even after appropriate adjustments for differences in sound speed between locations have been made. Here we present results from multiple Antarctic and European calibration events which suggest that echo-sounder transducer performance is dependant upon ambient water temperature. Highly significant differences in Sv and TS transducer gains were detected at both 38 and 120 kHz between calibrations conducted at the two locations. At 120 kHz, required Sv transducer gains were on average 1.4 dB less at South Georgia (sea temperature 2.0 °C) than in European waters (7.3 °C), and a similar trend was detected at 38 kHz. If European calibration parameters were to be employed on survey around South Georgia, and no account were taken of the differences in gain settings, then integrated 120 kHz echo signals would be under-reported by 2.8 dB, leading in turn to an under-estimation of krill biomass by 52.5 %. Every effort should therefore be made to ensure that echo-sounders are calibrated at a location as close as possible to the area in which surveys are to be conducted. In addition, the implications of temperature variation across a survey area for biomass estimation should be considered carefully.