We have previously shown that Lepeophtheirus salmonis produces trypsin and prostaglandin E(2) (PGE(2)) that are most likely responsible for the limited inflammatory response of Atlantic salmon to infection. After removal of the dopamine and PGE(2), the immunomodulatory activity of unfractionated and pools of the fractionated secretions was determined by examining the effects of the secretions on Atlantic salmon immune gene expression. Incubation of macrophage-enriched isolates of Atlantic salmon head kidney cells with the unfractionated secretion + PGE(2) revealed a significant inhibition of interleukin-1beta (IL-1beta) and major histocompatibility class I gene expression. Inhibition of lipopolysaccharide-induced IL-1beta expression in the Atlantic salmon head kidney cell line (SHK-1) was observed when three pools of the secretory/excretory products were tested. Further purification of products within these pools revealed that fraction 1-2 could account fully for the inhibition of IL-1beta expression in SHK-1 cells observed in pooled fraction 1. This study demonstrates that there are other immunomodulatory compounds produced by L. salmonis, in addition to PGE(2) and trypsin, that can inhibit the expression of Atlantic salmon immune-related genes in vitro.