The　effects　of　cobaltous　chloride,　methionine,　phosphate　and　fermentable　sugar　on　sisomicin　biosynthesis　in　a　batch　fermentation　process　were　investigated.　Fermentation　conditions　of　sisomicin　biosynthesis　in　a　batch　fermentation　process　were　optimized.　It　was　found　that　the　supplementation　with　some　components　in　medium　was　significant　to　sisomicin　production.　Methylation　of　precursor　gentamicin　A　in　sisomicin　biosynthesis　was　stimulated　by　6~10　mg/L　cobaltous　chloride　or　1.0~2.0　g/L　methionine.　The　best　time　of　methionine　supplementation　was　in　the　pre-middle　stage　of　sisomicin　biosynthesis.　High　phosphate　concentration　would　result　in　high　pyruvic　acid　concentration　and　amylase　activity,　and　low　alkaline　phosphatase　activity　and　sisomicin　concentration.　It　is　beneficial　to　sisomicin　biosynthesis　that　the　phosphate　concentration　is　controlled　under　3.14　mmol/L　in　cell　growth　phase　and　below　0.10　mmol/L　in　sisomicin　biosynthesis　phase.　Compared　with　that　in　the　batch　fermentation,　while　fermentable　sugar　was　controlled　in　8.5~11.5　g/L,　either　at　constant　or　varying　maltose　feeding　rate　or　at　constant　starch　hydrolysate　feeding　rate,　the　ferment　level　of　sisomicin　in　the　fed-batch　fermentations　was　distinctly　increased.