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The relationship between the immunological and biological activities of thyroid-stimulating hormone (TSH) isoforms present in the three human pituitary preparations 68/38 (1st IRP), 80/558 (2nd IRP) and 63/14 (MRC Research Standard A) was investigated. The isoforms were separated by chromatofocusing. Six peaks of immunoactivity were detected in 80/558, with pI values (means±S.E.M.) of 6·6±0·1, 6·2±0·1, 5·9±0·1, 5·5±0·1, 5·2±0·1 and 4·9±0·1. Four peaks, with pi values of 6·8±0·1, 5·9±0·1, 5·5±0·1 and 5·2±0·1, were observed for 68/38. Standard 63/14 had five peaks, with pI values of 6·9±0·1, 6·4±0·1, 5·9±0·1, 5·4±0·1 and 4·9±0·1. For each standard, six fractions around the peak areas and at the top and bottom of the gradient were pooled and microconcentrated to <1·0ml. Microconcentrated TSH samples were assayed in three TSH bioassays based upon FRTL-5 thyroid cells, utilizing cyclic AMP accumulation, iodide and thymidine uptake as end-points and standard 80/558 as reference preparation. The more acidic forms of TSH showed a higher biological: immunological (B: I) ratio for cyclic AMP accumulation with, for example, 63/14 having a maximum of 3·7 (pI 4·9) and a minimum of <0·7 (pI 6·9). In contrast, the maximum and minimum B: I ratios for iodide uptake for 63/14 were 3·8 (pI 6·9) and <0·8 (pI 4·6), and for thymidine uptake, maximum and minimum ratios were 7·2 (pI 6·9) and 1·1 (pI 4·6) respectively. In conclusion, the acidic forms of TSH stimulated cyclic AMP accumulation more than the alkaline forms, whereas the opposite was shown for iodide uptake and thymidine uptake.