The sequences coding for potential hemolytic sphingomyelinases (SMase) were found in five loci in Leptospira interrogans genome. These toxins are known as important factors to Leptospira infection and virulence. To evaluate their expression in Leptospira, we generated polyclonal antibodies against recombinant SMases (rSMases). The rSMases were obtained by heterologous expression in E. coli Bl 21 (DE3) Star pLysS. For expresion, three constructions were made in a plasmid with T7 based promoter, each one bearing the gene of a different SMase (Lic10657, Lic12631 and Lic11040 – the other two, Lic12632 and Lic3198 were not included in this study). The three toxins were expressed as inclusion body, and the purifications were carried out by nickel affinity chromatography. These toxins were weekly injected in BALB/c mices, and the serum was obtained in the fourth week. ELISA and western blot were done to confirm the titers and specificity of each antiserum. We observed a cross reaction between LIC10657 and LIC126310-antiserum and between LIC12631 and LIC10657-antiserum. This suggests that LIC10657 and LIC12631 have common epitopes, what may be related to a common ancestral sequence and/or common motifs.  To examine differences in SMase expression among Leptospira serovars, a western blot was done. A panel with 10 crude extracts of several Leptospira serovars was incubated with antiserum against the three rSMases. We only could detect expression of SMases in one sevorar: L. interrogans Pomona. In this serovar, we detected the expression of LIC10657 and LIC12631, but not LIC11040. The cross reaction between LIC10657 and LIC12631 was also observed in native toxins from Pomona extracts. The expression of these toxins just in one serovar may be related to differences in gene expression control. When we used Pomona culture constantly grown in culture media, the antiserum generated against LIC10657 recognized a band with the expected mass of LIC10657 leading us to suppose that it recognize the native LIC10657. But when we use Pomona that had recently infected hamsters, the band seem before are not present in the western blot and another band, about 10 kDa more than LIC0657, are observed. This band could be a modified LIC10657 or even an other related protein that can be recognized by anti-LIC10657. Nevertheless, this difference of expression, associated to environmental development, seems to be related to Leptospira infection and maintenance of virulence capacity.