The TSH releasing activity of two new analogues of TRH 'Pyr-(N3-Im-methyl)-His-Pro-NH-(n-amyl)' (I) and 'Pyr-His-Pro-(tyramine)' (II) was tested and compared with that of TRH in adult rats to test how structural modifications in the TRH molecule affect its biological activity: 1) in vitro in superfused pituitaries and 2) in vivo after ip injection, with measurement of TSH by RIA before and after addition of each secretagogue. Analogue I was found to be more potent than both TRH itself and Analogue II in stimulating TSH release: at 10 nmol/l in vitro, the ratio of induced to spontaneous release was 4.13 +/- 0.35, 2.98 +/- 0.20, and 1.19 +/- 0.17, respectively for each secretagogue, with a 50% effective dose of 6 X 10(-9) mol/l for Analogue I and 5 X 10(-8) mol/l for TRH. A similar order of potency in increasing plasma TSH (Analogue I greater than TRH greater than Analogue II) was found in vivo, as shown by dose-response curves. After a 4-day pre-treatment with TRH (2 X 100 micrograms/day) a similar TSH response to TRH and Analogue I (500 nmol/kg body weight) was observed. By contrast, the dose of Analogue II needed to obtain the same stimulatory effect on TSH release was twice as high. The biological activity of TRH appears to be more effectively increased by replacing an H atom by an amyl group in the C-terminal amide function of the proline residue of TRH than by a tyramyl group in the same residue.