Three new 8N-alkyl analogues of roseoflavin (MM), i.e., 8-ethylamino (EH), 8-methylethylamino (ME), 8-diethylamino-8-demethyl-D-riboflavin (EE), their tetraacetates, and 8-amino-8-demethyl-Driboflavin (HH) tetraacetate, were synthesized. A relation between the anti-riboflavin activity and the chemical structure of 8N-alkyl analogues (8N-methyl, ethyl) was studied by a restoration by riboflavin (RF) of inhibitory effect of the analogues on a growth of Grampositive bacteria, i.e., Sarcina lutea, Bacillus cereus, and Staphylococcus aureus. The inhibitory effect of most of the analogues was restored by RF. But in some cases, i.e., 8-methylamino-8-demethyl-D-riboflavin (MH) in Sar. lutea and MM in Staph. aureus, the effect was not completely restored. Apparently, the inhibition in early phase of growth was restored, but the maximum growth was still suppressed. The non-alkylated amino analogue (HH) showed only unrestorable suppression of maximum growth in Sar. lutea. Of restorable effect by RF of N-alkyl analogues, approximate decreasing orders of anti-RF activity were as follows. Dialkylated analogue>monoalkylated. HH showed insignificant anti-RF activity. In each group, methylated analogue>ethylated. In B. cereus monoalkylated analogues, and in Staph. aureus monoalkylated and EE showed no significant inhibitory effect. Redox potentials of the N-alkyl analogues were measured, and a definite relation between the chemical structure and the potential was found (RF=EE>ME>MM>>HH>EH>MH). But the anti-RF activity of the analogues was not completely explained by the difference of the redox potential from RF.