A rapid, accurate and non-invasive method to determine the sex of larval sea lampreys Petromyzon marinus, using wide-field pulse-scanning acoustic microscopy, is described. Crosssectional pulse-echo scans were made with a high-resolution acoustic microscope in 48 larvae (110-130 mm total length, L T ), and the acoustic images generated showed such internal body structures as the gonad, intestine, kidneys, cardinal veins, notochord and musculature. Females were identified by the presence of a relatively large (1-1Á5 mm diameter) ovary, which was considerably less reflective to the 15 to 25 MHz acoustic signals than the surrounding kidney tissue. Males were recognized by the lack of the large non-reflective ovary and, in some cases, the appearance of a small (0Á2-0Á3 mm) testis with slightly stronger reflective properties than the kidney. Identification of sex was confirmed by optical microscopy following dissection, and in a blind test on an additional 10 specimens (121-168 mm L T ), the acoustic method reliably identified sex in 100% of the larvae. These results indicate that acoustic microscopy can determine the sex of live sea lamprey larvae in c. 30 s per animal, a process which until now required dissection or invasive surgery. # 2004 The Fisheries Society of the British Isles