Interleukin-1beta (IL-1beta) is a central component in innate immunity and the inflammatory response of mammals. Only recently, the first non-mammalian IL-1beta sequences were published. In this study, we describe a second IL-1beta sequence (IL-1beta2) in carp with 74% amino acid identity to the carp IL-1beta1 sequence. The existence of two IL-1beta copies in the carp genome probably originates from the tetraploid nature of the species. In contrast to the first carp IL-1beta sequence, IL-1beta2 is represented by multiple genes with 95-99% identity. Detection of several IL-1beta2 sequences within individual homozygous fish suggests the presence of multiple copies of the IL-1beta2 gene in the carp genome, possibly as a result of subsequent gene duplication of IL-1beta2. In vivo, constitutive mRNA expression of both IL-1beta genes was found in healthy carp. IL-1beta2 mRNA expression could be up-regulated in head kidney cells similar to carp IL-1beta1, in vivo by infection with Trypanoplasma borreli and in vitro by stimulation with lipopolysaccharide (LPS). Cortisol, the major glucocorticoid in fish, is an endocrine-derived fator mediating IL-1beta expression. Although constitutive IL-1beta expression was inhibited by a physiological dose of cortisol, cortisol synergistically enhanced LPS-induced IL-1beta expression in carp. Involvement of the transcription factor nuclear factor (NF)-kappaB in expression of IL-1beta1 and IL-1beta2 was demonstrated. Ratio of IL-1beta expression was determined and this showed IL-1beta1 mRNA expression to be at least tenfold higher compared with IL-1beta2. The possibilities of IL-1beta2 being a functional gene or approaching pseudogene status are discussed.