Phytochemical examination of the fresh tubers of Brodiaea californica resulted in the isolation of four new steroidal saponins. Their structures were determined, by extensive spectral analysis including two-dimensional (2D) NMR spectroscopy and acid-catalyzed hydrolysis, to be (25S)-spirost-5-ene-1β, 3β-diol [(25S)-ruscogenin] 1-O-{O-β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside} (1), (25S)-ruscogenin 1-O-{O-β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranoside} (2), the C-20 and C-22 isomer of 2 (3) and the 6'-O-acetyl derivative of 2 (4), respectively. The conformations of the tetrasaccharide moiety of 2 and 4 were inspected through molecular mechanics and molecular dynamics calculation studies, showing that the acetyl group attached to C-6 of the inner glucose was near the C-21 methyl of the aglycon in the calculated preferred conformation of 4, which must cause the downfield shift of 21-Me by 0.07 ppm in comparing the ¹H-NMR of 4 with that of 2. The inhibitory activity of the isolated saponins on 12-O-tetradecanoylphorbor-13-acetate (TPA)-stimulated ³²P-incorporation into phospholipids of HeLa cells was evaluated to identify new antitumor-promoter compounds.