Again in the trio (5)
I believe the answer is:
three
'trio' is the definition.
(I know that trio can be written as three)
'again in the' is the wordplay.
'again' becomes 're' (prefix meaning 'again').
'in' indicates putting letters inside.
're' placed within 'the' is 'THREE'.
(Other definitions for three that I've seen before include "Snooker score for potting green ball" , "Number that's a crowd" , "Number constituting a crowd?" , "Bears or blind mice" , "Number of blind mice" .)