At least some mammalian tissues produce H2S in vitro from L-cysteine at rates sufficient to have physiological effects. To determine whether tissues of macrofaunal invertebrates have the same capacity, we measured H2S production in tissue homogenates of the Manila clam Tapes philippinarum and the lugworm Arenicola marina. Tissue homogenates from both animals produced significant quantities of H2S gas upon addition Of L-cysteine and the enzyme cofactor pyridoxal-? phosphate (10 mmol l(-1) and 2 mmol l(-1), respectively), while only tissues from T philippinarum produced measurable H2S in the absence of added substrate or cofactor. In T philippinarum tissues, H2S production was completely inhibited by the cystathionine beta-synthase (CBS) inhibitor aminooxyacetic acid (AOAA), suggesting that the majority of H2S production was via CBS pathways, while in A. marina body wall, AOAA inhibited only half of the total H2S production, indicating that the CBS pathway was not the only major source of H2S production. H2S production in tissues of T philippinarum but not A. marina was doubled by the addition of a second thiol substrate (2.5 mmol l(-1) 2-mercaptoethanol), suggesting the presence of an 'activated serine sulfhydrase pathway', which had previously been demonstrated only in some microfauna. (C) 2002 Elsevier Science Inc. All rights reserved.