Two Gram-positive-staining, rod-shaped, endospore-forming isolates (UG-2^T and UG-3), with an optimum growth temperature of around 37 °C and an optimum pH for growth of about 4, were recovered from an acidic effluent of the uranium mill tailing at Urgeiriça in Central Portugal. On the basis of 16S rRNA gene sequence similarity, the strains belonged to the family Sporolactobacillaceae and were closely related to Pullulanibacillus naganoensis ATCC 53909^T (97.9 %). Unlike P. naganoensis , strains UG-2^T and UG-3 grew in medium containing up to 5000 p.p.m. U(VI) but did not hydrolyse pullulan. Chemotaxonomic data also supported the affiliation of strains UG-2^T and UG-3 to the genus Pullulanibacillus . Physiological and biochemical tests along with fatty acid composition allowed differentiation of strains UG-2^T and UG-3 from P. naganoensis . It is suggested that strains UG-2^T and UG-3 represent a novel species, for which the name Pullulanibacillus uraniitolerans is proposed; the type strain is UG-2^T ( = DSM 19429^T = LMG 24205^T). An emended description of the genus Pullulanibacillus is also proposed.