The cyst wall of Colpoda steinii has been isolated and its chemical nature examined. It had a nitrogen content 13.9+/-0.2% (s.d.) and an ash 8.6+/-1.6% (s.d.). After lipid and hot-acid extraction there was a variable residual phosphorus of 0.19-0.64%. The protein nature, indicated by infrared and ultraviolet absorption, was confirmed when 100mug. of hydrolysed wall gave a ninhydrin colour equivalent to that given by 0.88-1.01mumoles of glycine. Hexosamine, hexose, pentose, lipid and dipicolinic acid were absent. 2. Paper chromatography of hydrolysates, besides showing the presence of the usual protein amino acids and three unidentified ninhydrin-reacting spots, indicated the presence of large amounts of glutamic acid. Estimated by chromatography, the amount present was 52.9+/-0.6 (s.d.) g./100g. of ash-free wall; manometric estimation of l-glutamic acid with l-glutamate 1-carboxy-lyase gave 46.5+/-0.9 (s.d.) g./100g. 3. Free carboxyl groups were estimated by titration as 0.159+/-0.011 (s.d.) mole/100g. and those present as amide as 0.154+/-0.004 (s.d.) mole/100g., and the total was compared with the dicarboxylic acid content 0.360+/-0.010 (s.d.) mole/100g. 4. After treatment with 98% formic acid 25-30% of the wall material could be extracted by 0.05m-sodium carbonate solution (extract 1); after treatment of the residue with performic acid a further 62-63% based on the original weight could be extracted by 0.05m-sodium carbonate (extract 2). 5. The average values found for the glutamic acid contents were 21.7g./100g. for extract 1 and 58.0g./100g. for extract 2. The cysteic acid content of whole oxidized wall was about 5.8g./100g. and of extract 2 also about 5.8g./100g. The glutamic acid and cysteic acid contents of the final residue were also investigated. 6. The significance of these extraction experiments in relation to the wall structure is discussed.
